Dale R. Abrahamson

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.

Mouse stem cells seeded into decellularized rat kidney scaffolds endothelialize and remodel basement membranes.

Introduction To address transplant organ shortage, a promising strategy is to decellularize kidneys in a manner that the scaffold retains signals for seeded pluripotent precursor cells to differentiate and recapitulate native structures: matrix-to-cell signaling followed by cell-cell and cell-matrix interactions, thereby remodeling and replacing the original matrix. This would reduce scaffold antigenicity and enable xeno-allografts. Results DAPI-labeled cells in arterial vessels and glomeruli were positive for both endothelial lineage markers, BsLB4 and VEGFR2. Rat scaffold’s basement membrane demonstrated immunolabeling with anti-mouse laminin β1. Labeling intensified over time with 14 day incubations. Conclusion We provide new evidence for matrix-to-cell signaling in acellular whole organ scaffolds that induces differentiation of pluripotent precursor cells to endothelial lineage. Production of mouse basement membrane supports remodeling of host (rat)-derived scaffolds and thereby warrants further investigation as a promising approach for xenotransplantation. Methods We previously showed that murine embryonic stem cells arterially seeded into acellular rat whole kidney scaffolds multiply and demonstrate morphologic, immunohistochemical and gene expression evidence for differentiation. Vascular cell endothelialization was now further tested by endothelial specific BsLB4 lectin and anti-VEGFR2 (Flk1) antibodies. Remodeling of the matrix basement membranes from rat to mouse (“murinization”) was assessed by a monoclonal antibody specific for mouse laminin β1 chain.

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.

β1 integrin is necessary for ureteric bud branching morphogenesis and maintenance of collecting duct structural integrity

The kidney collecting system develops from branching morphogenesis of the ureteric bud (UB). This process requires signaling by growth factors such as glial cell line derived neurotrophic factor (GDNF) and fibroblast growth factors (FGFs) as well as cell extracellular matrix interactions mediated by integrins. The importance of integrin signaling in UB development was investigated by deleting integrin β1 at initiation (E10.5) and late (E18.5) stages of development. Deletion at E10.5 resulted in a severe branching morphogenesis phenotype. Deletion at E18.5 did not alter renal development but predisposed the collecting system to severe injury following ureteric obstruction. β1 integrin was required for renal tubular epithelial cells to mediate GDNF- and FGF-dependent signaling despite normal receptor localization and activation in vitro. Aberrations in the same signaling molecules were present in the β1-null UBs in vivo. Thus β1 integrins can regulate organ branching morphogenesis during development by mediating growth-factor-dependent signaling in addition to their well-defined role as adhesion receptors.

Laminin cleavage by activated human neutrophils yields proteolytic fragments with selective migratory properties

We studied the interactions between human neutrophils, as well as the purified human neutrophil serine proteases elastase (HNE) and cathepsin G (HNCG), and laminin. Our results show that intact laminin and two proteolytic fragments generated by HNE bind to neutrophils and stimulate cell migration. Domain‐ specific antilaminin monoclonal antibodies, rotary shadowing electron microscopy, and Western blotting mapped the two promigratory fragments on the laminin cross to the apical three‐armed region and long arm, respectively. In contrast, a fragment derived from the terminal ends of short arms neither bound to neutrophils nor stimulated migration. When neutrophils embedded in a reconstituted basement membrane gel were activated with phorbol myristate acetate, several stable, proteolytic laminin fragments were released into supernatants. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and Western blotting showed that these fragments appeared identical to those generated after digestion of soluble laminin with HNE and HNCG. Furthermore, release of laminin fragments by embedded neutrophils was inhibited by diisopropyl fluorophos‐ phate, and duplicated by incubating the basement membrane gel with purified HNE and HNCG. Our findings therefore suggest that neutrophils, through release of HNE and HNCG, are capable of digesting basement membrane laminin in vivo. In addition, the release of laminin fragments from damaged basement membranes may promote neutrophil migration and thereby accelerate inflammatory processes.

Loss of α3/α4(IV) Collagen from the Glomerular Basement Membrane Induces a Strain-Dependent Isoform Switch to α5α6(IV) Collagen Associated with Longer Renal Survival in Col4a3−/− Alport Mice

Mutations in COL4A3/4/5 genes that affect the normal assembly of the α3/4/5(IV) collagen network in the glomerular basement membrane (GBM) cause Alport syndrome. Patients progress to renal failure at variable rates that are determined by the underlying mutation and putative modifier genes. Col4a3 −/− mice, a model for autosomal recessive Alport syndrome, progress to renal failure significantly slower on the C57BL/6 than on the 129X1/Sv background. Reported here is a novel strain-specific alternative collagen IV isoform switch that is associated with the differential renal survival in Col4a3 −/− Alport mice. The downregulation or the absence of α3/4(IV) collagen chains in the GBM of Lmx1b −/− and Col4a3 −/− mice was found to induce ectopic deposition of α5/6(IV) collagen. The GBM deposition of α5/6(IV) collagen was abundant in C57BL/6 Col4a3 −/− mice but almost undetectable in 129X1/Sv Col4a3 −/− mice. This strain difference was due to overall low expression of α6(IV) chain and α5/6(IV) protomers in the tissues of 129X1/SvJ mice, a natural Col4a6 knockdown. In (129 × B6)F1 Col4a3 −/− mice, the amount of α5/6(IV) collagen in the GBM was inherited in a mother-to-son manner, suggesting that it is controlled by one or more X-linked loci, possibly Col4a6 itself. Importantly, high levels of ectopic α5/6(IV) collagen in the GBM were associated with approximately 46% longer renal survival. These findings suggest that α5/6(IV) collagen, the biologic role of which has been hitherto unknown, may partially substitute for α3/4/5(IV) collagen. Therapeutically induced GBM deposition of α5/6(IV) collagen may provide a novel strategy for delaying renal failure in patients with autosomal recessive Alport syndrome.