Patricia L. St. John

Integration of Embryonic Stem Cells in Metanephric Kidney Organ Culture

Many stages of nephrogenesis can be studied using cultured embryonic kidneys, but there is no efficient technique available to readily knockdown or overexpress transgenes for rapid evaluation of resulting phenotypes. Embryonic stem (ES) cells have unlimited developmental potential and can be manipulated at the molecular genetic level by a variety of methods. The aim of this study was to determine if ES cells could respond to developmental signals within the mouse embryonic day 12 to embryonic day 13 (E12 to E13) kidney microenvironment and incorporate into kidney structures. ROSA26 ES cells were shown to express beta-galactosidase ubiquitously when cultured in the presence of leukemia inhibitory factor to suppress differentiation. When these cells were microinjected into E12 to E13 metanephroi and then placed in transwell organ culture, ES cell-derived, beta-galactosidase-positive cells were identified in epithelial structures resembling tubules. On rare occasions, individual ES cells were observed in structures resembling glomerular tufts. Electron microscopy showed that the ES cell-derived tubules were surrounded by basement membrane and had apical microvilli and junctional complexes. Marker analysis revealed that a subset of these epithelial tubules bound Lotus tetragonolobus and expressed alpha(1) Na(+)/K(+) ATPase. ES cells were infected before injection with a cytomegalovirus promoter-green fluorescence protein (GFP) adenovirus and GFP expression was found as early as 18 h, persisting for up to 48 h in cultured kidneys. This ES cell technology may achieve the objective of obtaining a versatile cell culture system in which molecular interventions can be used in vitro and consequences of these perturbations on the normal kidney development program in vivo can be studied.

Cellular Origins of Type IV Collagen Networks in Developing Glomeruli

Laminin and type IV collagen composition of the glomerular basement membrane changes during glomerular development and maturation. Although it is known that both glomerular endothelial cells and podocytes produce different laminin isoforms at the appropriate stages of development, the cellular origins for the different type IV collagen heterotrimers that appear during development are unknown. Here, immunoelectron microscopy demonstrated that endothelial cells, mesangial cells, and podocytes of immature glomeruli synthesize collagen alpha 1 alpha 2 alpha1(IV). However, intracellular labeling revealed that podocytes, but not endothelial or mesangial cells, contain collagen alpha 3 alpha 4 alpha 5(IV). To evaluate the origins of collagen IV further, we transplanted embryonic kidneys from Col4a3-null mutants (Alport mice) into kidneys of newborn, wildtype mice. Hybrid glomeruli within grafts containing numerous host-derived, wildtype endothelial cells never expressed collagen alpha 3 alpha 4 alpha 5(IV). Finally, confocal microscopy of glomeruli from infant Alport mice that had been dually labeled with anti-collagen alpha 5(IV) and the podocyte marker anti-GLEPP1 showed immunolabeling exclusively within podocytes. Together, these results indicate that collagen alpha 3 alpha 4 alpha 5(IV) originates solely from podocytes; therefore, glomerular Alport disease is a genetic defect that manifests specifically within this cell type.

Direct visualization of renal vascular morphogenesis inFlk1 heterozygous mutant mice

Flk1, a receptor tyrosine kinase for vascular endothelial growth factor (VEGF), is the earliest known marker for endothelial precursors (angioblasts). We examined heterozygous mice in which the Flk1gene was partially replaced by a promoter-less LacZ insert and used β-galactosidase histochemistry to view cells transcribing Flk1. In day 10 ( E10) embryos, a Flk1-positive network surrounded the metanephric blastema, and, at E11, a vessel entered the metanephros from its ventral aspect alongside the ingrowing ureteric bud. However, aortic branches did not engage embryonic kidneys at these time points. In newborns, β-galactosidase was localized exclusively and intensely to endothelial cells of all vessels and glomeruli. In contrast, when E12 kidneys grown in organ culture for 6 days were examined, only scattered Flk1-positive cells were seen, glomeruli were unlabeled, and vessels were absent. When organ-cultured kidneys were then grafted into wild-type anterior eye chambers, numerous Flk1-positive endothelial cells in vessels and glomeruli were found, all stemming from the graft. Image analysis showed that grafts with the most abundant glomerulo- and tubulogenesis were also those with the richest expression of Flk1. We conclude that 1) kidney microvessels precede renal artery development, 2) angioblast differentiation is arrested in organ culture but released on grafting when vasculogenesis resumes, and 3) nephrogenesis and microvessel assembly are tightly coupled in vivo.

Early embryonic renal tubules of wild-type and polycystic kidney disease kidneys respond to cAMP stimulation with cystic fibrosis transmembrane conductance regulator/Na(+),K(+),2Cl(-) Co-transporter-dependent cystic dilation.

Metanephric organ culture has been used to determine whether embryonic kidney tubules can be stimulated by cAMP to form cysts. Under basal culture conditions, wild-type kidneys from embryonic day 13.5 to 15.5 mice grow in size and continue ureteric bud branching and tubule formation over a 4- to 5-d period. Treatment of these kidneys with 8-Br-cAMP or the cAMP agonist forskolin induced the formation of dilated tubules within 1 h, which enlarged over several days and resulted in dramatically expanded cyst-like structures of proximal tubule and collecting duct origin. Tubule dilation was reversible upon withdrawal of 8-Br-cAMP and was inhibited by the cAMP-dependent protein kinase inhibitor H89 and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTR(inh)172. For further testing of the role of CFTR, metanephric cultures were prepared from mice with a targeted mutation of the Cftr gene. In contrast to kidneys from wild-type mice, those from Cftr -/- mice showed no evidence of tubular dilation in response to 8-Br-cAMP, indicating that CFTR Cl(-) channels are functional in embryonic kidneys and are required for cAMP-driven tubule expansion. A requirement for transepithelial Cl(-) transport was demonstrated by inhibiting the basolateral Na(+),K(+),2Cl(-) co-transporter with bumetanide, which effectively blocked all cAMP-stimulated tubular dilation. For determination of whether cystic dilation occurs to a greater extent in PKD kidneys in response to cAMP, Pkd1(m1Bei) -/- embryonic kidneys were treated with 8-Br-cAMP and were found to form rapidly CFTR- and Na(+),K(+),2Cl(-) co-transporter-dependent cysts that were three- to six-fold larger than those of wild-type kidneys. These results suggest that cAMP can stimulate fluid secretion early in renal tubule development during the time when renal cysts first appear in PKD kidneys and that PKD-deficient renal tubules are predisposed to abnormally increased cyst expansion in response to elevated levels of cAMP.

A Mutant Receptor Tyrosine Phosphatase, CD148, Causes Defects in Vascular Development

ABSTRACT Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPη), participates in developmental vascularization. A mutant allele, CD148ΔCyGFP, was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions.

Podocyte Expression of Hypoxia-Inducible Factor (HIF)–1 and HIF–2 during Glomerular Development

The heterodimeric transcription factors, hypoxia-inducible factor (HIF)-1 and HIF-2, are essential for the maintenance of cellular oxygen homeostasis. In response to hypoxia, stabilized HIF-1alpha and HIF-2alpha proteins bind HIF-1beta and initiate expression of genes that alleviate hypoxic stress, including those promoting neovascularization. Both HIF-1 and HIF-2 stimulate transcription of vascular endothelial growth factor (VEGF), a crucial regulator of vascular development. Because VEGF is highly expressed by metanephric podocytes and collecting ducts, developing mouse kidney was examined for the presence and distribution of HIF-1alpha, HIF-2alpha, and HIF-1beta. The expression of HIF-1alpha and HIF-2alpha mRNAs in newborn mouse kidney was confirmed by RT-PCR and Northern blot analysis. By in situ hybridization, HIF-1alpha and HIF-2alpha mRNAs were highly expressed in the nephrogenic zone of newborn kidney cortex and in the medulla. Particularly intense hybridization was found in podocytes of developing glomeruli and in medullary collecting ducts. Both HIF-1 and HIF-2 heterodimers were identified in newborn kidney lysates by immunoprecipitation with HIF-1alpha, HIF-2alpha, and HIF-1beta antibodies and Western blots. Immunofluorescence analysis of the hypoxia marker, pimonidazole, showed that collecting ducts and many developing tubules undergo severe hypoxia in developing kidney. Immunohistochemistry of newborn kidney demonstrated widespread expression of HIF-1beta protein in nuclei of glomeruli and all tubular segments, whereas HIF-2alpha protein expression was more restricted and localized chiefly to podocytes of developing glomeruli and developing tubules. HIF-1alpha and HIF-2alpha protein and VEGF mRNA were all strongly induced in embryonic kidneys maintained in hypoxic organ cultures. Collectively, these data suggest that HIF stabilization, by hypoxia and/or by other means, may be critical for VEGF production and kidney vascular development.

Laminin Compensation in Collagen α3(IV) Knockout (Alport) Glomeruli Contributes to Permeability Defects

Alport disease is caused by mutations in genes encoding the alpha3, alpha4, or alpha5 chains of type IV collagen, which form the collagenous network of mature glomerular basement membrane (GBM). In the absence of alpha3, alpha4, alpha5 (IV) collagen, alpha1, alpha2 (IV) collagen persists, which ordinarily is found only in GBM of developing kidney. In addition to dysregulation of collagen IV, Alport GBM contains aberrant laminins, which may contribute to the progressive GBM thickening and splitting, proteinuria, and renal failure seen in this disorder. This study sought to characterize further the laminin dysregulation in collagen alpha3(IV) knockout mice, a model of Alport disease. With the use of confocal microscopy, laminin alpha1 and alpha5 abundance was quantified, and it was found that they co-distributed in significantly large amounts in areas of GBM thickening. In addition, labeling of entire glomeruli for laminin alpha5 was significantly greater in Alport mice than in wild-type siblings. Reverse transcriptase-PCR from isolated glomeruli demonstrated significantly more laminin alpha5 mRNA in Alport mice than in wild-type controls, indicating upregulated transcription of Lama5. For testing glomerular barrier function, ferritin was injected into 2-wk-old Alport and control mice, and GBM was examined by electron microscopy. Highest ferritin levels were seen in Alport GBM thickenings beneath effaced podocyte foot processes, but morphologically normal GBM was significantly permeable as well. We concluded that (1) ultrastructurally normal Alport GBM residing beneath differentiated podocyte foot processes is inherently and abnormally permeable, and (2) upregulation of Lama5 transcription and concentration of laminin alpha1 and alpha5 within Alport GBM thickenings contribute to abnormal permeabilities.

Partial Rescue of Glomerular Laminin 5 Mutations by Wild-Type Endothelia Produce Hybrid Glomeruli

Both endothelial cells and podocytes are sources for laminin alpha1 at the inception of glomerulogenesis and then for laminin alpha5 during glomerular maturation. Why glomerular basement membranes (GBM) undergo laminin transitions is unknown, but this may dictate glomerular morphogenesis. In mice that genetically lack laminin alpha5, laminin alpha5beta2gamma1 is not assembled, vascularized glomeruli fail to form, and animals die at midgestation with neural tube closure and placental deficits. It was previously shown that renal cortices of newborn mice contain endothelial progenitors (angioblasts) and that when embryonic day 12 kidneys are transplanted into newborn kidney, hybrid glomeruli (host-derived endothelium and donor-derived podocytes) result. Reasoning that host endothelium may correct the glomerular phenotype that is seen in laminin alpha5 mutants, alpha5 null embryonic day 12 metanephroi were grafted into wild-type newborn kidney. Hybrid glomeruli were identified in grafts by expression of a host-specific LacZ lineage marker. Labeling of glomerular hybrid GBM with chain-specific antibodies showed a markedly stratified distribution of laminins: alpha5 was found only on the inner endothelial half of GBM, whereas alpha1 located to outer layers beneath mutant podocytes. For measurement of the contribution of host endothelium to hybrid GBM, immunofluorescent signals for laminin alpha5 were quantified: Hybrid GBM contained approximately 50% the normal alpha5 complement as wild-type GBM. Electron microscopy of glomerular hybrids showed vascularization, but podocyte foot processes were absent. It was concluded that (1) endothelial and podocyte-derived laminins remain tethered to their cellular origin, (2) developing endothelial cells contribute large amounts of GBM laminins, and (3) podocyte foot process differentiation may require direct exposure to laminin alpha5.

Binding of Injected Laminin to Developing Kidney Glomerular Mesangial Matrices and Basement Membranes In Vivo

During glomerular development, subendothelial and -epithelial basement membrane layers fuse to produce the glomerular basement membrane (GBM) shared by endothelial cells and epithelial podocytes. As glomeruli mature, additional basement membrane derived from podocytes is spliced into the fused GBM and loose mesangial matrices condense. The mechanisms for GBM fusion, splicing, and mesangial matrix condensation are not known but might involve intermolecular bond formation between matrix molecules. To test for laminin binding sites, we intravenously injected mouse laminin containing α1-, β1-, and γ1-chains into 2-day-old rats. Kidneys were immunolabeled for fluorescence and electron microscopy with domain-specific rat anti-mouse laminin monoclonal antibodies (MAbs), which recognized only mouse and not endogenous rat laminin. Intense labeling for injected laminin was found in mesangial matrices and weaker labeling was seen in GBMs of maturing glomeruli. These patterns persisted for at least 2 weeks after injection. In control newborns receiving sheep IgG, no binding of injected protein was observed and laminin did not bind adult rat glomeruli. To assess which molecular domains might mediate binding to immature glomeruli, three proteolytic laminin fragments were affinity-isolated by MAbs and injected into newborns. These failed to bind glomeruli, presumably owing to enzymatic digestion of binding domains. Alternatively, stable incorporation may require multivalent laminin binding. We conclude that laminin binding sites are transiently present in developing glomeruli and may be functionally important for GBM assembly and mesangial matrix condensation.

Permeability of the macula densa basement membrane area to high molecular weight molecules.

To investigate the permeability properties of the basement membrane beneath macula densa cells and between extraglomerular mesangial cells, thick ascending limbs with attached glomeruli were dissected from rabbit kidney and incubated in a Ringers solution containing either horseradish peroxidase (HRP) (1 mg/ml; molecular weight approximately 40,000) or native or cationic ferritin (both at 5 mg/ml molecular weight approximately 450,000) for 5 and 20 min at room temperature. Tubules were processed for electron microscopy. At both time points, HRP reaction product fully permeated the matrix material between extraglomerular mesangial cells, the basement membrane underneath the macula densa, and also was occasionally located in intercellular spaces and intracellular vesicles within macula densa cells. Similar results were obtained with native and cationic ferritin. In separate experiments, thick ascending limbs with attached glomeruli were perfused for 20 min at room temperature with a Ringer solution containing HRP (1 mg/ml). HRP was found in tubulovesicular bodies within the apical cytoplasm of macula densa cells again indicating that these cells exhibit endocytotic activity. However, HRP did not gain access to intercellular spaces indicating that the apical junctional complex was impermeable to HRP. These results demonstrate that the macula densa basement membrane and matrix material between extraglomerular mesangial cells is permeable to high molecular weight molecules and suggest unhindered diffusion of water and solutes within this area.