Endostatin regulates branching morphogenesis of renal epithelial cells and ureteric bud.

Endostatin (ES) inhibits endothelial cell migration and has been found to bind to glypicans (Gpcs) on both endothelial cells and renal epithelial cells. We examined the possibility that ES might regulate epithelial cell morphogenesis. The addition of ES to cultured epithelial cells causes an inhibition of both hepatocyte growth factor- and epidermal growth factor-dependent process formation and migration. In contrast, ES does not inhibit epidermal growth factor-dependent morphogenesis in renal epithelial cells derived from Gpc-3 -/mice, whereas expression of Gpc-1 in these cells reconstitutes ES responsiveness. Gpc-3 -/mice have been shown to display enhanced ureteric bud (UB) branching early in development, and cultured UB cells release ES into the media, suggesting that ES binding to Gpcs may regulate UB branching. The addition of ES inhibits branching of the explanted UB, whereas a neutralizing Ab to ES enhances UB outgrowth and branching. Thus, local expression of ES at the tips of the UB may play a role in the regulation of UB arborization.

Glypicans and the biology of renal malformations.

The molecular mechanisms that control renal development are largely undefined. The discovery of mutations in the gene encoding glypican-3 (Gpc3) in humans with Simpson-Golabi-Behmel syndrome (SGBS) and renal dysplasia, and the establishment of a genetic mouse model of GPC3 deficiency has provided an opportunity to define the role of GPC3 during renal development. Glypicans are a family of cell surface heparan sulfate proteoglycans that control growth factor signalling in nonrenal tissues. Mutational inactivation of Gpc3 causes somatic overgrowth and cystic renal dysplasia, as observed in SGBS. Overgrowth of the ureteric bud and its branches and increased ureteric bud cell proliferation is observed during the early stages of renal development. Subsequently, during corticomedullary differentiation, cortical collecting duct cell proliferation is increased, while medullary collecting duct cells proliferate at a reduced rate and undergo apoptosis resulting in degeneration of the medulla. However, cells that constitute medullary cysts are characterized by enhanced cell proliferation and a lower rate of apoptosis. Thus, the phenotype arising from Gpc3 inactivation demonstrates that tight regulation of cell proliferation and apoptosis is critical during formation of the renal medulla.

Glypican-3 modulates BMP- and FGF-mediated effects during renal branching morphogenesis.

The kidney of the Gpc3-/ mouse, a novel model of human renal dysplasia, is characterized by selective degeneration of medullary collecting ducts preceded by enhanced cell proliferation and overgrowth during branching morphogenesis. Here, we identify cellular and molecular mechanisms underlying this renal dysplasia. Glypican-3 (GPC3) deficiency was associated with abnormal and contrasting rates of proliferation and apoptosis in cortical (CCD) and medullary collecting duct (MCD) cells. In CCD, cell proliferation was increased threefold. In MCD, apoptosis was increased 16-fold. Expression of Gpc3 mRNA in ureteric bud and collecting duct cells suggested that GPC3 can exert direct effects in these cells. Indeed, GPC3 deficiency abrogated the inhibitory activity of BMP2 on branch formation in embryonic kidney explants, converted BMP7-dependent inhibition to stimulation, and enhanced the stimulatory effects of KGF. Similar comparative differences were found in collecting duct cell lines derived from GPC3-deficient and wild type mice and induced to form tubular progenitors in vitro, suggesting that GPC3 directly controls collecting duct cell responses. We propose that GPC3 modulates the actions of stimulatory and inhibitory growth factors during branching morphogenesis.

BMP7 controls collecting tubule cell proliferation and apoptosis via Smad1-dependent and -independent pathways.

Bone morphogenetic protein-7 (BMP7) controls ureteric bud and collecting duct morphogenesis in a dose-dependent manner (Piscione TD, Yager TD, Gupta IR, Grinfeld B, Pei Y, Attisono L, Wrana JL, and Rosenblum ND. Am J Physiol Renal Physiol 273: F961-F975, 1997). We defined cellular and molecular mechanisms underlying these effects in embryonic kidney explants and in the mIMCD-3 cell model of collecting tubule morphogenesis. Low-dose (0.25 nM) BMP7 significantly increased tubule number and cell proliferation. Similar to BMP2, high-dose (10 nM) BMP7 inhibited cell proliferation and stimulated apoptosis. To define molecular mechanisms, we identified signaling events downstream of BMP7. High-dose BMP7, but not low-dose BMP7, activated Smad1 in mIMCD-3 cells. Moreover, the inhibitory effects of high-dose BMP7 and BMP2, but not the stimulatory effects of low-dose BMP7, on tubulogenesis and cell proliferation were significantly reduced in mIMCD-3 cells stably expressing Smad1(Delta458), a dominant negative mutant form of Smad1, but not in cells stably expressing wild-type Smad1. We conclude that BMP7 exerts dose-dependent effects on ureteric bud or collecting duct cell proliferation and apoptosis by signaling via Smad1-dependent and Smad1-independent pathways.

Vesicoureteric reflux associated with renal dysplasia in the Wolf-Hirschhorn syndrome.

Wolf-Hirschhorn syndrome (WHS) is caused by a partial deletion of the short arm of chromosome 4 (4p16.3) and is characterized by severe pre- and postnatal growth retardation, developmental delay, and multiple congenital anomalies, including malformations of the urogenital system. We describe the renal and urinary tract phenotype in a series of six children with WHS. Vesicoureteric reflux was present in four of our six patients (5 of 10 ureters), an abnormality not previously reported in WHS.

BMP-2/ALK3 and HGF signal in parallel to regulate renal collecting duct morphogenesis.

Bone morphogenetic protein (BMP)-2 and hepatocyte growth factor (HGF) exert antagonistic effects on renal collecting duct formation during embryogenesis. A current model proposes HGF inhibits BMP-2 signaling at the level of Smad1 in a common target cell. Here, we show that BMP-2 and HGF control collecting duct formation via parallel pathways. We examined the interactions between BMP-2 and HGF in the mIMCD-3 model of collecting duct morphogenesis. During tubule formation, HGF rescued the inhibitory effects of BMP-2 and of a constitutive active form of the BMP-2 receptor, ALK3, stably expressed in mIMCD-3 cells. To determine whether the effect of HGF occurs through known mediators which act downstream of the BMP-2/ALK3 complex, we examined the effect of HGF on BMP-2-induced Smad1 phosphorylation, Smad1/Smad4 complex formation, and Smad1 nuclear translocation. Neither HGF nor other receptor tyrosine kinase ligands (EGF, FGF-4) induced phosphorylation of endogenous Smad1 in mIMCD-3 cells or in Mv1Lu, MC3T3-E1 or P19 cells. Furthermore, none of these ligands blocked induction of the BMP-responsive promoter, Tlx2. Thus, HGF overcomes the inhibitory effects of BMP-2 on collecting duct morphogenesis without interrupting any of the known signaling events in the BMP-2 dependent Smad1 signaling pathway. We conclude that BMP-2/ALK3 and HGF function to control parallel pathways downstream of their respective cell surface receptors. Integration of these signals likely occurs at the level of transcriptional or post-transcriptional events.

Protein kinase A is a negative regulator of renal branching morphogenesis and modulates inhibitory and stimulatory bone morphogenetic proteins.

Protein kinase A (PKA) regulates morphogenetic responses to bone morphogenetic proteins (BMPs) during embryogenesis. However, the mechanisms by which PKA regulates BMP function are unknown. During kidney development, BMP-2 and high doses of BMP-7 inhibit branching morphogenesis, whereas low doses of BMP-7 are stimulatory (Piscione, T. D., Yager, T. D., Gupta, I. R., Grinfeld, B., Pei, Y., Attisano, L., Wrana, J. L., and Rosenblum, N. D. (1997) Am. J. Physiol. 273, F961-F975). We examined the interactions between PKA and these BMPs in embryonic kidney explants and in the mouse inner medullary collecting duct-3 model of collecting duct morphogenesis. H-89, an inhibitor of PKA, stimulated branching morphogenesis and enhanced the stimulatory effect of low doses of BMP-7 on tubule formation. Furthermore, H-89 rescued the inhibition of tubulogenesis by BMP-2 (or high doses of BMP-7) by attenuating BMP-2-induced collecting duct apoptosis. In contrast, 8-bromo-cAMP, an activator of PKA, inhibited tubule formation and attenuated the stimulatory effects of low doses of BMP-7. To determine mechanisms underlying the interdependence of BMP signaling and PKA activity, we examined the effect of PKA on the known signaling events in the BMP-2-dependent Smad1 signaling pathway and the effect of BMP-2 on PKA activity. PKA did not induce endogenous Smad1 phosphorylation, Smad1-Smad4 complex formation, or Smad1 nuclear translocation. In contrast, BMP-2 increased endogenous PKA activity and induced phosphorylation of the PKA effector, cAMP-response element-binding protein, in a PKA-dependent manner. We conclude that BMP-2 induces activation of PKA and that PKA regulates the effects of BMPs on collecting duct morphogenesis without activating the known signaling events in the BMP-2-dependent Smad1 signaling pathway.

Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome.

Glypicans are a family of heparan sulfate proteoglycans that are linked to the cell surface through a glycosyl-phosphatidylinositol anchor. One member of this family, glypican-3 (Gpc3), is mutated in patients with the Simpson-Golabi-Behmel syndrome (SGBS). These patients display pre- and postnatal overgrowth, and a varying range of dysmorphisms. The clinical features of SGBS are very similar to the more extensively studied Beckwith-Wiedemann syndrome (BWS). Since BWS has been associated with biallelic expression of insulin-like growth factor II (IGF-II), it has been proposed that GPC3 is a negative regulator of IGF-II. However, there is still no biochemical evidence indicating that GPC3 plays such a role.Here, we report that GPC3-deficient mice exhibit several of the clinical features observed in SGBS patients, including developmental overgrowth, perinatal death, cystic and dyplastic kidneys, and abnormal lung development. A proportion of the mutant mice also display mandibular hypoplasia and an imperforate vagina. In the particular case of the kidney, we demonstrate that there is an early and persistent developmental abnormality of the ureteric bud/collecting system due to increased proliferation of cells in this tissue element. The degree of developmental overgrowth of the GPC3-deficient mice is similar to that of mice deficient in IGF receptor type 2 (IGF2R), a well characterized negative regulator of IGF-II. Unlike the IGF2R-deficient mice, however, the levels of IGF-II in GPC3 knockouts are similar to those of the normal littermates.

The malformed kidney: disruption of glomerular and tubular development.

Renal malformations are the major cause of renal failure during early childhood. They are found in approximately 100 genetic syndromes. We review the embryologic development of the kidney and its molecular control. Important new information has been derived from mutational analysis in humans and mice. We describe how mutations in nine transcription factors, 12 signaling molecules and nine gene products involved in a variety of other cellular functions disrupt renal morphogenesis. The information presented provides a template for integrating new discoveries on the molecular basis of renal development, for classifying renal malformations observed in the clinical setting, and for identifying defective genes in affected patients.

BMP-2 and OP-1 exert direct and opposite effects on renal branching morphogenesis.

The bone morphogenetic proteins, BMP-2 and OP-1, are candidates for growth factors that control renal branching morphogenesis. We examined their effects in embryonic kidney explants and in the mIMCD-3 cell model of collecting duct morphogenesis (mIMCD-3 cells are derived from the terminal inner medullary collecting duct of the SV40 mouse). Osteogenic protein-1 (OP-1), at a dose of 0.25 nM, increased explant growth by 30% (P = 0.001). In contrast, 100-fold greater concentrations of OP-1 (28 nM) decreased explant growth by 10% (P < 0.001). BMP-2 was entirely inhibitory (maximum inhibition of 7% at 5 nM, P < 0.0004). In an in vitro model for branching morphogenesis utilizing the kidney epithelial cell line, mIMCD-3, low doses of OP-1 (< 0.5 nM) increased the number of tubular structures formed by 28 +/- 5% (P = 0.01), whereas concentrations > 0.5 nM decreased that number by 22 +/- 8% (P = 0.02). All concentrations of BMP-2 (0.05-10 nM) were inhibitory (maximum inhibition at 10 nM of 88 +/- 3%, P < 0.0001). Stimulatory doses of OP-1 increased tubular length (P = 0.003) and the number of branch points/structure (3.2-fold increase, P = 0.0005) compared with BMP-2. To determine the molecular basis for these effects, we demonstrated that BMP-2 is bound to mIMCD-3 cells by the type I serine/threonine kinase receptor, ALK-3, and that OP-1 bound to an approximately 80-kDa protein using ligand-receptor affinity assays. To demonstrate that OP-1 can exert both stimulatory and inhibitory effects within a developing kidney, embryonic explants were treated with agarose beads saturated with 2 microM OP-1. OP-1 decreased the number of ureteric bud/collecting duct branches adjacent to the beads by 58 +/- 1% (P < 0.0001). In contrast, the number of branches in tissue distal to the OP-1 beads was enhanced, suggesting a stimulatory effect at lower doses of OP-1. We conclude that OP-1 and BMP-2 directly control branching morphogenesis and that the effects of OP-1 are dependent on its local concentration within developing kidney tissue.

Recombinant alpha 1(VIII) collagen chains form homotrimers in vitro.

Type VIII collagen, a member of the short chain collagen family, is expressed in large blood vessels and in the subendothelium and mesangium of the glomerulus. While two genetically distinct type VIII collagen chains, alpha 1 and alpha 2, have been identified, it is not known whether type VIII collagen exists as homotrimers or heterotrimers. To determine whether alpha 1(VIII) collagen chains can associate to form homotrimers, a full length human alpha 1(VIII) collagen cDNA was generated by overlap extension PCR and used as a substrate for coupled in vitro transcription/translation. A translation product of 80kDa, the predicted size of alpha 1(VIII) collagen, was identified by autoradiography of SDS-gels containing radiolabelled translation products. Sensitivity of the in vitro translated protein to digestion with bacterial collagenase and trypsin and the size of the proteins generated by these digestions provide evidence that alpha 1(VIII) collagen can participate in the formation of homotrimers.

The pedagogic characteristics of a clinical conference for senior residents and faculty.

OBJECTIVE: To determine the pedagogic characteristics of a clinical conference for senior pediatric residents and selected faculty. PARTICIPANTS AND SETTING: Nineteen senior pediatric residents and 14 selected faculty members participated in a daily clinical conference at Children's Hospital, Boston, Mass. DESIGN: Qualitative research design using videotapes of nine consecutive hour-long sessions to generate pedagogic topics to be investigated using a questionnaire administered to participating residents and faculty. Narrative responses were analyzed to find pedagogic themes. RESULTS: Analysis of videotapes generated the following three topics: What facilitated learning? What was learned? What makes the process of teaching and learning effective? In the questionnaire residents indicated that learning was facilitated by resident-faculty interactions (19/19), faculty participation (19/19), and information resources (12/19). Content learned included information (16/19), approach to diagnosis (11/19), management strategies (14/19), and different perspective (14/19). An effective process of teaching and learning was attributed to case-based resident initiated discussion (19/19), facilitation by the chief resident (16/19), and non-competitive discussions in which expert faculty played a nondominant role (19/19). Faculty identified identical factors relating to all three themes. The mean rating of the conference was 4.5/5 (SD, +/- 0.50) and 4.7/5 (SD, +/- 0.45) by residents and faculty, respectively (Likert scale, 1 to 5). CONCLUSIONS: The pedagogic effectiveness of this conference was attributed to a resident-centered, case-based learning format and a discussion process characterized by noncompetitive interactions among faculty and residents, strong group facilitation by the chief resident, and participation of faculty experts in a nondominant role.

Expression of a Xenopus counterpart of mammalian syndecan 2 during embryogenesis.

We have identified a Xenopus cDNA, XS-2, by screening a Xenopus embryonic stage-22-24 cDNA library with a DNA probe encoding the transmembrane and cytoplasmic domains of mouse syndecan 1. The 1.4 kb cDNA consists of an open reading frame of 642 nucleotides encoding a protein of 191 amino acids. The predicted protein of 20869 Da contains a 25-amino acid putative transmembrane domain and a 32-amino acid putative cytoplasmic domain, both of which are highly similar to the corresponding regions of rat syndecan 2 (92% identity) and to a lesser degree those of rat syndecans 1, 3 and 4 (62, 64 and 78% respectively). The putative N-terminal ectodomain contains a possible attachment site for heparan sulphate, identical with the comparable glycosaminoglycan-attachment sequence of rat syndecan 2. Polyclonal antisera raised against recombinant ectodomain of XS-2, expressed as a fusion protein, recognized a heparan sulphate proteoglycan in XTC cell-culture medium. This proteoglycan bound to DEAE-Sephacel and was eluted with 1 M NaCl; digestion with heparitinase but not chondroitinase ABC resulted in the identification of a 46 kDa protein by these antisera. Northern-blot analysis indicated that XS-2 identifies two Xenopus mRNA species approx. 4 and 2 kb in size in embryos ranging in maturation from the 64-cell stage to stage 54. These results demonstrate that a heparan sulphate proteoglycan, similar to syndecan 2, is expressed during Xenopus embryogenesis.

Predicting medical student success in a clinical clerkship by rating students' nonverbal behavior.

OBJECTIVE: To evaluate the relative contribution of affective and cognitive skills to ratings of students' clinical performance by their supervisors. METHODS: Each students' nonverbal behavior was analyzed by examining 33 nonverbal behavioral characteristics in videotape of each of 36 students interviewing a parent or patient three times during a 4-week pediatric clerkship. These ratings were then compared with the student's formal academic evaluation. RESULTS: The 33 nonverbal behavioral characteristics rated for each student were reduced to five composite variables. Three of these correlated significantly with the final grade, providing an affective profile of the highly rated student. Regression analysis of the five composite variables revealed that affective skills accounted for at least 46% of the variance in the students' final grades (multiple R = .68, P = .0015). CONCLUSIONS: Ratings of students' affective characteristics were highly related to clinical evaluations in this pediatric setting. Students who were evaluated highly had a specific affective profile that could be described by analysis of their nonverbal behaviors.

The mesangial matrix in the normal and sclerotic glomerulus.

Mesangial sclerosis is a final common pathway to glomerular destruction in a variety of glomerular diseases. The expression of several classes of extracellular matrix (ECM) molecules has been defined in the normal and diseased mesangial matrix (MM). However, the manner in which these ECM components determine the three dimensional structure and function of the MM remains to be defined. Structural studies of the MM suggest that its constituent molecules are regionally organized into subcompartments with different three dimensional structures. The diversity of matrix molecules expressed within the MM as well as the organization of these components in nonrenal ECM's, such as the cornea, provides further support for this organizational model. The study of the cornea has also revealed that novel short chain collagenous proteins partially determine the three dimensional structure of the matrix. Recently, a novel collagen, type VIII collagen, has been described in mesangial cells and in the intact glomerulus. It is hypothesized that type VIII collagen is expressed both as a polymer and as a monomer within the glomerulus, and depending on its conformation, may serve unique functions. In the chronically diseased MM, normal MM components are overexpressed and fibrillar collagens are expressed de novo in a delayed fashion. Enhanced proteoglycan expression, observed early in disease, may determine increased volume of the mesangium. This, in turn, may stimulate the production of fibrillar collagens by mesangial cells resulting in a fibrillar noncompliant mesangial matrix.

Alpha 1-VIII collagen is expressed in the rat glomerulus and in resident glomerular cells.

Current knowledge regarding the molecular composition of extracellular matrices in the glomerulus does not explain how these components interact to form stable three-dimensional structures. The recent recognition that short-chain collagens such as type VIII collagen function as molecular bridges in some nonrenal tissues has raised the possibility that such molecules may serve a similar function in the glomerulus. We have recently shown that cultured rat mesangial cells synthesize and secrete several short-chain collagenous proteins, one of which has properties similar to alpha 1-VIII collagen. In the present study we have isolated a rat mesangial cell alpha 1-VIII collagen cDNA clone, the sequence of which is 81% homologous to mouse alpha 1-VIII collagen. We used this cDNA to determine that alpha 1-VIII collagen mRNA is expressed in rat renal cortex and in cultured glomerular mesangial, epithelial, and endothelial cells. Additionally, we demonstrated that alpha 1-VIII collagen is secreted by cultured mesangial cells as an 80-kDa translation product. By immunocytochemistry, alpha 1-VIII collagen localized to the media of large intrarenal arteries and to the capillary loops and the mesangium of normal rat kidney. These results indicate that type VIII collagen is a normal constituent of the rat glomerulus as well as large intrarenal arteries. We speculate that type VIII collagen may function in part to determine the three-dimensional organization of the subendothelial and mesangial matrices.

Site-specific expression of collagen I and XII mRNAs in the rat periodontal ligament at two developmental stages.

In mammals, the periodontal ligament (PDL) is a highly specialized tissue which facilitates tooth eruption and lends mechanical support to the tooth once in occlusion. The PDL extracellular matrix fibers play a major role in such functions. During its development, the spatial arrangement of the PDL extracellular matrix undergoes rapid changes. So that it could be determined whether the structural alteration in the PDL is associated with changes in the expression of collagenous proteins with different functional properties, the transcriptional patterns of collagens I and XII were examined. The maxillary dento-alveolar segments, each containing three molars, from 25-day-old and 40-day-old Sprague-Dawley rats were selected as being representative of developing and matured tissues, respectively. Rat alpha 2(I) collagen cDNA and rat alpha 1(XII) collagen cDNA were used as molecular probes for identification of the corresponding mRNAs by RNA transfer blot analysis, RNase protection assay, and in situ hybridization. The results showed that alpha 2(I) collagen mRNA was expressed in both developing and matured tissues. However, the level of expression decreased with maturity. In contrast, the expression of alpha 1(XII) collagen was increased in the matured tissue as compared with the developing tissue. In situ hybridization in these tissues indicated that the expression of alpha 1(XII) collagen mRNA was limited to the mature stage of PDL development. It is suggested that collagen fibril arrangement during PDL development may be related to the expression of collagen XII.