Mouse Metanephric Mesenchymal Cell-Derived Angioblasts Undergo Vasculogenesis in Three-Dimensional Culture.

In vitro models for the investigation of renal vascular development are limited. We previously showed that isolated metanephric mesenchymal (MM) and ureteric bud (UB) cells grown in three-dimensional (3D) matrices formed organoids that consisted of primitive vascular structures surrounding a polarized epithelium. Here, we examined the potential of two principal effectors of vasculogenesis, vascular endothelial growth factor A (VEGF-A), and platelet-derived growth factor B chain (PDGF-BB), to stimulate MM cell differentiation. The results showed that MM cells possess angioblast characteristics by expressing phenotypic markers for endothelial and mesenchymal cells. UB cells synthesize VEGF-A and PDGF-BB proteins and RNA, whereas the MM cells express the respective cognate receptors, supporting their role in directional induction of vasculogenesis. VEGF-A stimulated proliferation of MM cells in monolayer and in 3D sponges but did not affect MM cell migration, organization, or vasculogenesis. However, PDGF-BB stimulated MM cell proliferation, migration, and vasculogenesis in monolayer and organization of the cells into primitive capillary-like assemblies in 3D sea sponge scaffolds in vitro. A role for PDGF-BB in vasculogenesis in the 3D MM/UB co-culture system was validated by direct interference with PDGF-BB or PDGF receptor-ß cell interactions to implicate PDGF-BB as a primary effector of MM cell vasculogenesis. Thus, MM cells resemble early renal angioblasts that may provide an ideal platform for the investigation of renal vasculogenesis in vitro.

Prophylactic eculizumab for kidney transplantation in a child with atypical hemolytic uremic syndrome due to complement factor H mutation.

We present a case of successful deceased-donor kidney transplantation in a three-yr-old child with aHUS due to complement factor H mutation, using only prophylactic eculizumab treatment prior to transplant. She developed disease exacerbation in the immediate post-operative period despite having therapeutic eculizumab concentrations and evidence for complete complement pathway blockade. The patient responded well to additional doses of eculizumab and has maintained excellent graft function and disease control in the first year post-transplantation. The optimal dosing scheme for eculizumab in the perioperative period remains to be determined. More sensitive biomarkers of early disease activity are needed to improve disease monitoring. Finally, the duration of eculizumab therapy in patients with aHUS remains to be determined.

Reciprocal induction of simple organogenesis by mouse kidney progenitor cells in three-dimensional co-culture.

Kidney development is regulated by a coordinated reciprocal induction of metanephric mesenchymal (MM) and ureteric bud (UB) cells. Here, established MM and UB progenitor cell lines were recombined in three-dimensional Matrigel implants in SCID mice. Differentiation potential was examined for changes in phenotype, organization, and the presence of specialized proteins using immunofluorescence and bright-field and electron microscopy. Both cell types, when grown alone, did not develop into specialized structures. When combined, the cells organized into simple organoid structures of polarized epithelia with lumens surrounded by capillary-like structures. Tracker experiments indicated the UB cells formed the tubuloid structures, and the MM cells were the source of the capillary-like cells. The epithelial cells stained positive for pancytokeratin, the junctional complex protein ZO-1, collagen type IV, as well as UB and collecting duct markers, rearranged during transfection (RET), Dolichos biflorus lectin, EndoA cytokeratin, and aquaporin 2. The surrounding cells expressed α-smooth muscle actin, vimentin, platelet endothelial cell adhesion molecule 1 (PECAM), and aquaporin 1, a marker of vasculogenesis. The epithelium exhibited apical vacuoles, microvilli, junctional complexes, and linear basement membranes. Capillary-like structures showed endothelial features with occasional pericytes. UB cell epithelialization was augmented in the presence of MM cell-derived conditioned medium, glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), or fibronectin. MM cells grown in the presence of UB-derived conditioned medium failed to undergo differentiation. However, UB cell-derived conditioned medium induced MM cell migration. These studies indicate that tubulogenesis and vasculogenesis can be partially recapitulated by recombining individual MM and UB cell lineages, providing a new model system to study organogenesis ex vivo.

Erratum: Deficiency of complement factor H-related proteins and autoantibody-positive hemolytic uremic syndrome in an infant with combined partial deficiencies and autoantibodies to complement factor H and ADAMTS13.

[This corrects the article DOI: 10.1093/ckj/sfy010.].

Mitogenic signaling via platelet-derived growth factor beta in metanephric mesenchymal cells.

Mice deficient in either platelet-derived growth factor (PDGF) B chain or PDGF receptor (PDGFR) beta lack mesangial cells. PDGF stimulates proliferation and migration of metanephric mesenchymal cells, from which mesangial cells are derived. Binding of PDGF to PDGFR-beta induces autophosphorylation at specific tyrosine residues and activates various effector proteins, including phosphatidylinositol-3-kinase (PI3-K). This study explored the role of PI 3-K and reactive oxygen species (ROS) in PDGF-mediated signaling using cells established from wild-type and PDGFR-beta -/- metanephric blastemas at 11.5 days post-conception. PDGF-induced effects that were dependent on PI3-K activation were determined using PDGFR-beta -/- cells made to express "add-back" mutant PDGFR-beta capable of binding PI3-K. We found that PDGF is mitogenic for mesenchymal cells expressing PDGFR-beta, and PI3-K is an important regulator of PDGF-induced DNA synthesis. Activation of ERK1/2 is partially dependent on PI3-K, and both the PI3-K and MEK-ERK1/2 pathways contribute to PI3-K-dependent mitogenesis. In addition, PDGF-induced DNA synthesis in wild-type cells was found to be dependent on ROS that are generated downstream of PI3-K activation. Using antisense oligonucleotides and small interfering RNA, we determined that the NAD(P)H oxidase Nox4 produces these ROS that activate Akt and the MEK-ERK1/2 mitogenic cascade. In conclusion, the present study demonstrates Nox4 involvement in PDGF-induced DNA synthesis in metanephric mesenchymal cells and provides the first evidence that PDGF-induced PI3-K activity enhances production of ROS by Nox4.

Deficiency of complement factor H-related proteins and autoantibody-positive hemolytic uremic syndrome in an infant with combined partial deficiencies and autoantibodies to complement factor H and ADAMTS13.

A 3-month-old male infant developed an extremely severe episode of atypical hemolytic uremic syndrome (aHUS) associated with partial deficiencies of full-length complement factor H (FH; ∼15% of infant normal) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) (39% of normal) and autoantibodies reactive with both proteins. His FH and ADAMTS13 genes were normal, indicating that the partial deficiencies were acquired, probably as the result of autoantibodies against full-length FH and ADAMTS13. The child also had a homozygous deletion of the complement factor H-related (CFHR)3-CFHR1 portion in the complement factor H (CFH) gene cluster. He therefore had deficiency of CFHR proteins and autoantibody-positive hemolytic uremic syndrome (DEAP-HUS) with an unusual early onset associated with a partial deficiency of ADAMTS13 and an anti-ADAMTS13 autoantibody. His clinical episode of aHUS responded to plasma infusion and subsequent treatment with mycophenolate and rituximab. We believe that this is the first report of DEAP-HUS in an infant with partial deficiencies in both ADAMTS13 and full-length FH acquired in association with autoantibodies to both proteins.

Ethical issues associated with conducting genetic family studies of complex disease.

PURPOSE: To examine subjects' recognition of the risks and ethical issues associated with enrollment in genetic family studies (GFS) and explore how this recognition affects their informed and voluntary participation. METHODS: A cross-sectional study design including both quantitative and qualitative data was employed. Structured interviews using the Contextual Assessment Approach Questionnaire (CAA-Q) were conducted with 246 Mexican American (MA) participants. To gain in-depth understanding of questionnaire responses, semi-structured interviews with 30 participants were conducted. All participants were interviewed before their enrollment in the Family Investigation of Nephropathy and Diabetes (FIND). RESULTS: Subjects' average age was 56 years; 62% were females. Seventy-eight percent of participants were not formally educated beyond high school and 72% reported an annual household income of < or =20,000 dollars. Eighty-five percent agreed to provide researchers with information on relatives' ages, gender, and education. Sixty-five percent of participants were willing to provide identifiable information such as names, addresses, and phone numbers of relatives. Sixty-three percent of participants indicated that there were direct benefits (i.e., supporting research) to disclosing relatives' information. Seventy-six percent stated that there were no risks associated with participation in GFS (e.g., discrimination or confidentiality of genetic information) compared with 10% who said that there were such risks. While discussing potential risks, subjects did not consider these to influence their decision to participate. CONCLUSIONS: Subjects enrolled in GFS did not recognize and tended to underestimate the social and cultural risks associated with their participation in GFS. If subjects do not fully comprehend the risks, this raises questions concerning their ability to provide informed consent and to voluntarily participate. We propose a subject-centered approach that views enrollment as an active process in which subjects and recruiters give and receive information on risks and ethical issues related to participation, which enhances protection of the rights and welfare of subjects participating in GFS.

Morphological insights into the origin of glomerular endothelial and mesangial cells and their precursors.

Glomerular endothelial and mesangial cells may originate from the metanephric mesenchyme. We used the MAb Thy1.1, a mesangial cell marker in the adult rat kidney, and rat endothelial cell markers MAb RECA-1, MAb PECAM-1 (CD31), and MAb Flk-1 as potential markers to characterize the spatial and temporal distribution of mesangial and endothelial cell precursors during nephrogenesis in the rat. At early stages of glomerulogenesis, RECA-1- and Thy1.1-positive cells were detected in the metanephric blastema at 14 days post conception (dpc) embryos and 15 dpc, respectively, with Thy1.1 expression in cells surrounding the ureteric bud. At 17 and 18 dpc, both RECA-1- and Thy1.1-positive cells were found in the cleft of the S-shaped bodies and in the capillary loops of maturing glomeruli. Double staining for BrdU, a marker of proliferation, and for RECA-1 or BrdU and Thy1.1 also localize in the cleft of S-shaped bodies and in glomerular capillary loops at later stages of development. PDGFRbeta co-localizes in cells expressing endothelial or mesangial markers. The data suggest that endothelial and mesangial cell precursors share common markers during the course of glomerulogenesis and that full differentiation of these cells occurs at late stages of glomerular maturation. Thy1.1- and RECA-1-positive cells may be derived from the metanephric blastemal cells at early stages of kidney development. A subpopulation of these Thy1.1- or RECA-1-positive cells may be precursors that can migrate into the cleft of comma and S-shaped bodies and proliferate in situ to form glomerular capillary tufts.

Direct and efficient cellular transformation of primary rat mesenchymal precursor cells by KSHV.

Infections by viruses are associated with approximately 12% of human cancer. Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several malignancies commonly found in AIDS patients. The mechanism of KSHV-induced oncogenesis remains elusive, due in part to the lack of an adequate experimental system for cellular transformation of primary cells. Here, we report efficient infection and cellular transformation of primary rat embryonic metanephric mesenchymal precursor cells (MM cells) by KSHV. Cellular transformation occurred at as early as day 4 after infection and in nearly all infected cells. Transformed cells expressed hallmark vascular endothelial, lymphatic endothelial, and mesenchymal markers and efficiently induced tumors in nude mice. KSHV established latent infection in MM cells, and lytic induction resulted in low levels of detectable infectious virions despite robust expression of lytic genes. Most KSHV-induced tumor cells were in a latent state, although a few showed heterogeneous expression of lytic genes. This efficient system for KSHV cellular transformation of primary cells might facilitate the study of growth deregulation mechanisms resulting from KSHV infections.

PDGF receptor-{beta} modulates metanephric mesenchyme chemotaxis induced by PDGF AA.

PDGF B chain or PDGF receptor (PDGFR)-beta-deficient (-/-) mice lack mesangial cells. To study responses of alpha- and beta-receptor activation to PDGF ligands, metanephric mesenchymal cells (MMCs) were established from embryonic day E11.5 wild-type (+/+) and -/- mouse embryos. PDGF BB stimulated cell migration in +/+ cells, whereas PDGF AA did not. Conversely, PDGF AA was chemotactic for -/- MMCs. The mechanism by which PDGFR-beta inhibited AA-induced migration was investigated. PDGF BB, but not PDGF AA, increased intracellular Ca(2+) and the production of reactive oxygen species (ROS) in +/+ cells. Transfection of -/- MMCs with the wild-type beta-receptor restored cell migration and ROS generation in response to PDGF BB and inhibited AA-induced migration. Inhibition of Ca(2+) signaling facilitated PDGF AA-induced chemotaxis in the wild-type cells. The antioxidant N-acetyl-l-cysteine (NAC) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI) abolished the BB-induced increase in intracellular Ca(2+) concentration, suggesting that ROS act as upstream mediators of Ca(2+) in suppressing PDGF AA-induced migration. These data indicate that ROS and Ca(2+) generated by active PDGFR-beta play an essential role in suppressing PDGF AA-induced migration in +/+ MMCs. During kidney development, PDGFR beta-mediated ROS generation and Ca(2+) influx suppress PDGF AA-induced chemotaxis in metanephric mesenchyme.

Darbepoetin alfa for the treatment of anemia in pediatric patients with chronic kidney disease.

Darbepoetin alfa, an erythropoiesis-stimulating glycoprotein, has proved efficacious in the treatment of anemia of chronic kidney disease (CKD) in adult subjects. However, little information is available from pediatric populations. We conducted an open-label, non-inferiority, 28-week study comparing the efficacy of darbepoetin alfa with that of recombinant human erythropoietin (rHuEpo) in pediatric subjects with CKD. Subjects, aged 1-18, who were receiving stable rHuEpo treatment (n=124) were randomized (1:2) to either continue receiving rHuEpo or convert to darbepoetin alfa, with doses titrated to achieve and maintain hemoglobin (Hb) levels between 10.0 and 12.5 g/dl. Darbepoetin alfa was considered to be non-inferior to rHuEpo if the lower limit of the two-sided 95% confidence interval (CI) for the difference in the mean change in Hb between the two treatment groups was above -1.0 g/dl. The adjusted mean change in Hb between the baseline and the evaluation period for the rHuEpo and darbepoetin alfa groups was -0.16 g/dl and 0.15 g/dl, respectively, with a difference of 0.31 g/dl (95% CI: -0.45, 1.07) between the means. These results, and the comparable safety profiles, demonstrate that darbepoetin alfa is non-inferior to rHuEpo in the treatment of anemia in pediatric patients with CKD.

Effect of platelet-derived growth factor isoforms in rat metanephric mesenchymal cells.

Platelet-derived growth factor (PDGF) B-chain or PDGF beta-receptor-deficient mice lack mesangial cells. To explore potential mechanisms for failure of PDGF A-chain to rescue mesangial cell phenotype, we investigated the biological effects and signaling pathways of PDGF AA and PDGF BB in metanephric mesenchymal (MM) cells isolated from rat kidney. PDGF AA caused modest cell migration but had no effect on DNA synthesis, unlike PDGF BB, which potently stimulated migration and DNA synthesis. PDGF AA and PDGF BB significantly increased the activities of phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase (MAPK). PDGF BB was more potent than PDGF AA in activating PI 3-K or MAPK in these cells. Pretreatment of MM cells with the MAPK kinase (MEK) inhibitor PD-098059 abrogated PDGF BB-induced DNA synthesis, whereas the PI 3-K inhibitor wortmannin had a very modest inhibitory effect on DNA synthesis (approximately Delta20%). On the other hand, wortmannin completely blocked PDGF AA- and PDGF BB-induced migration, whereas PD-098059 had a modest inhibitory effect on cell migration. These data demonstrate that activation of MAPK is necessary for the mitogenic effect of PDGF BB, whereas PI 3-K is required for the chemotactic effect of PDGF AA and PDGF BB. Although PDGF AA stimulates PI 3-K and MAPK activity, it is not mitogenic and only modestly chemotactic. Collectively, the data may have implications related to the failure of PDGF AA to rescue mesangial cell phenotype in PDGF B-chain or PDGF-beta-receptor deficiency.

Cloning of the 5'-flanking region of the murine bone morphogenetic protein-7 gene.

BMP-7, a member of the bone morphogenetic protein subfamily of the TGFbeta-superfamily is highly expressed in the murine kidney. BMP-7 is involved in fetal nephron development and mesenchymal to epithelial cell differentiation. Constitutive BMP-7 expression is found in tubular and glomerular epithelial cells of the adult kidney. BMP-7 may play a role in physiology and pathophysiology of the adult kidney since BMP-7 gene expression in acute renal ischemia is diminished and injection of recombinant BMP-7 into rats with ischemic acute renal failure preserves renal function. In order to investigate the transcriptional regulation of BMP-7, this study was undertaken to clone and characterize the promoter of the murine BMP-7 gene. A 1394 bp sequence of the 5'-flanking region of the BMP-7 gene was isolated and subcloned. No TATA and CAAT box consensus motifs could be identified as shown for promoters of other BMPs. Using in vitro transfection assays, the 5'-flanking region revealed moderate to strong basal promoter activity. PMA increased basal BMP-7 promoter activity. Thus BMP-7 gene transcription might involve at least in part a PKC-dependent pathway. The cloning of a 5'-flanking region of the BMP-7 gene should provide a useful tool for future studies on the transcriptional regulation of BMP-7 gene expression.

Incorporating the Contextual Assessment Approach to regimens used in genetic family studies.

PURPOSE: To develop a procedure that enhances enrollment and addresses ethical issues associated with participation in genetic family studies. METHODS: The Contextual Assessment Approach (CAA) was standardized to the recruitment procedures in the Family Investigation of Nephropathy and Diabetes (FIND) study at the University of Texas Health Science Center at San Antonio. Structured interviews with the CAA questionnaire (CAA-Q) were conducted with 50 low-income Mexican-American probands. The CAA allows systematic interpretation of health beliefs, family dynamics, and attitudes regarding participation in FIND. Data analyses included qualitative and quantitative methods. RESULTS: CAA analyses of probands' perspectives regarding relatives' enrollment in FIND facilitated recruiting 34 probands from whom 30 families were enrolled (family enrollment rate: 88%). CAA reduced recruitment efforts by 32% and avoided exerting undue pressure on unwilling participants to ensure voluntary participation. Remarkably, 76% of the subjects were unaware of any risk associated with participation in genetic family studies. CONCLUSIONS: Administering the CAA-Q before enrolling subjects in FIND increased our enrollment rate by targeting efforts toward the willing subjects and addressing ethical issues associated with their participation.

Fibronectin induces ureteric bud cells branching and cellular cord and tubule formation.

BACKGROUND: The extracellular matrix (ECM) protein fibronectin is involved in several stages of embryogenesis. Fibronectin exerts its effect through interaction with cellular integrin and nonintegrin receptors. METHODS: We investigated the effect of fibronectin on branching and tubulogenesis of ureteric bud cells in a three-dimensional gel culture system. Primary ureteric bud cells from mouse embryos at gestation 11 days (E11) were isolated and established in culture. Fibronectin and integrin subunits were localized using immunoperoxidase staining. RESULTS: In three-dimensional collagen type I gel culture of ureteric bud cell, fibronectin dose dependently induces cord and tubule formation. Both ureteric bud cells and ureteric bud branches in embryonic kidney express the same multiple integrin subunits that include beta(1), beta(3), alpha(3), alpha(4) and alpha(v). Embryonic kidneys examined at E12, E14, and E16 days of gestation express fibronectin in the undifferentiated mesenchyme especially next to ureteric bud branches and in the interstitium around glomerulotubular structures and blood vessels. Fibronectin expression was similar at the tips and stalks of branching ureteric bud. Fibronectin expression is maximum at E12 and decreases with advanced gestation. Cultured ureteric bud cells also express fibronectin. RGD peptides inhibit cord and tubular formation in the three-dimensional gel. Anti-alpha(3)beta(1) antibody partially inhibits fibronectin-induced cord and tubule formation. Hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and glial cell line-derived neurotrophic factor (GDNF) induce ureteric bud cell cord formation in three-dimensional gel. The effects of growth factors are delayed and quantitatively less compared to the effect of fibronectin. CONCLUSION: Fibronectin induces ureteric bud cells branching and tubulogenesis through interaction with multiple integrin receptors. Cultured ureteric bud cells express fibronectin and the origin of fibronectin at mesenchyme-ureteric bud interface is likely both the metanephric mesenchyme and ureteric bud epithelium. Addition of individual neutralizing antibodies to beta(1), beta(3), alpha(3), alpha(4,)alpha(6) and alpha(v) integrin subunits does not block the effect of fibronectin. Only an antibody to alpha(3)beta(1) integrin substantially blocks the effect of fibronectin. Other mechanisms, including unidentified integrins, are likely involved in fibronectin-induced cord and tubule formation.