Kathryn Isom

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.

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.

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.

Deletion of Von Hippel-Lindau in Glomerular Podocytes Results in Glomerular Basement Membrane Thickening, Ectopic Subepithelial Deposition of Collagen α1α2α1(IV), Expression of Neuroglobin, and Proteinuria

Vascular endothelial growth factor, which is critical for blood vessel formation, is regulated by hypoxia inducible transcription factors (HIFs). A component of the E3 ubiquitin ligase complex, von Hippel-Lindau (VHL) facilitates oxygen-dependent polyubiquitination and proteasomal degradation of HIFalpha subunits. Hypothesizing that deletion of podocyte VHL would result in HIFalpha hyperstabilization, we crossed podocin promoter-Cre transgenic mice, which express Cre recombinase in podocytes beginning at the capillary loop stage of glomerular development, with floxed VHL mice. Vascular patterning and glomerular development appeared unaltered in progeny lacking podocyte VHL. However, urinalysis showed increased albumin excretion by 4 weeks when compared with wild-type littermates with several sever cases (>1000 microg/ml). Many glomerular ultrastructural changes were seen in mutants, including focal subendothelial delamination and widespread podocyte foot process broadening, and glomerular basement membranes (GBMs) were significantly thicker in 16-week-old mutants compared with controls. Moreover, immunoelectron microscopy showed ectopic deposition of collagen alpha1alpha2alpha1(IV) in GBM humps beneath podocytes. Significant increases in the number of Ki-67-positive mesangial cells were also found, but glomerular WT1 expression was significantly decreased, signifying podocyte death and/or de-differentiation. Indeed, expression profiling of mutant glomeruli suggested a negative regulatory feedback loop involving the HIFalpha prolyl hydroxylase, Egln3. In addition, the brain oxygen-binding protein, Neuroglobin, was induced in mutant podocytes. We conclude that podocyte VHL is required for normal maintenance of podocytes, GBM composition and ultrastructure, and glomerular barrier properties.

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.

Kidney development and gene expression in the HIF2α knockout mouse

The hypoxia‐inducible transcription factor‐2 (HIF2), a heterodimer composed of HIF2α and HIF1β subunits, drives expression of genes essential for vascularization, including vascular endothelial growth factor (VEGF) and VEGF receptor‐2 (VEGFR‐2, Flk‐1). Here, we used a HIF2α/LacZ transgenic mouse to define patterns of HIF2α transcription during kidney development and maturation. Our results from embryonic heterozygotes showed HIF2α/LacZ expression by apparently all renal endothelial cells. At 4 weeks of age, glomerular mesangial and vascular smooth muscle cells were also positive together with endothelial cells. These expression patterns were confirmed by electron microscopy using Bluo‐gal as a β‐galactosidase substrate. Small numbers of glomerular and tubular epithelial cells were also positive at all stages examined. Light and electron microscopic examination of kidneys from HIF2α null embryos showed no defects in renal vascular development or nephrogenesis. Similarly, the same amounts of Flk‐1 protein were seen on Western blots of kidney extracts from homozygous and heterozygous HIF2α mutants. To examine responsiveness of HIF2α null kidneys to hypoxia, embryonic day 13.5 metanephroi were cultured in room air or in mild (5% O2) hypoxia. For both heterozygous and null samples, VEGF mRNA levels doubled when metanephroi were cultured in mild hypoxia. Anterior chamber grafts of embryonic HIF2α knockouts were morphologically indistinguishable from heterozygous grafts. Endothelial markers, platelet endothelial cell adhesion molecule and BsLB4, as well as glomerular epithelial markers, GLEPP1 and WT‐1, were all expressed appropriately. Finally, we undertook quantitative real‐time polymerase chain reaction of kidneys from HIF2α null embryos and wild‐type siblings and found no compensatory up‐regulation of HIF1α or ‐3α. Our results show that, although HIF2α was widely transcribed by kidney endothelium and vascular smooth muscle, knockouts displayed no detectable deficits in vessel development or VEGF or Flk‐1 expression. Developmental Dynamics 236:1115–1125, 2007.

Developmental expression of a mucinlike glycoprotein (MUCLIN) in pancreas and small intestine of CF mice

The mucinlike glycoprotein MUCLIN, one of two protein products of the CRP-ductin gene, was used to study changes in the expression of sulfated glycoconjugates during the pathogenesis of cystic fibrosis, using the cystic fibrosis transmembrane conductance regulator (CFTR) knockout mouse (CF mouse). We assessed the appearance of dilated lumina containing protein or mucus plugs in pancreatic acini and crypts of the small intestine and quantified MUCLIN protein and CRP-ductin mRNA during postnatal development. In CF mice, the pancreatic acinar lumen was dilated by postnatal day 16 (P16), but MUCLIN protein was first significantly increased by P23 and remained elevated through adulthood compared with normal mice. Similarly, intestinal crypts had CF-like mucus plugs by P16, but MUCLIN protein was first elevated by P23 and remained elevated through adulthood compared with normal mice. In both organs, MUCLIN labeling of the luminal surface was increased concomitantly with dilation and protein or mucus plugging but before upregulation of expression. The morphological changes were then followed by upregulation of MUCLIN protein and CRP-ductin mRNA expression. This is the first direct study of CF pathogenesis and the resultant increase in glycoconjugate gene expression. The data are consistent with CF pathogenesis progressing from an initial alteration in protein secretory dynamics (increased luminal MUCLIN and protein/mucus plugs) to an upregulation of glycoprotein/mucin gene expression, which is expected to exacerbate obstruction of the luminal spaces.The mucinlike glycoprotein MUCLIN, one of two protein products of the CRP-ductin gene, was used to study changes in the expression of sulfated glycoconjugates during the pathogenesis of cystic fibrosis, using the cystic fibrosis transmembrane conductance regulator (CFTR) knockout mouse (CF mouse). We assessed the appearance of dilated lumina containing protein or mucus plugs in pancreatic acini and crypts of the small intestine and quantified MUCLIN protein and CRP-ductin mRNA during postnatal development. In CF mice, the pancreatic acinar lumen was dilated by postnatal day 16( P16), but MUCLIN protein was first significantly increased by P23 and remained elevated through adulthood compared with normal mice. Similarly, intestinal crypts had CF-like mucus plugs by P16, but MUCLIN protein was first elevated by P23 and remained elevated through adulthood compared with normal mice. In both organs, MUCLIN labeling of the luminal surface was increased concomitantly with dilation and protein or mucus plugging but before upregulation of expression. The morphological changes were then followed by upregulation of MUCLIN protein and CRP-ductin mRNA expression. This is the first direct study of CF pathogenesis and the resultant increase in glycoconjugate gene expression. The data are consistent with CF pathogenesis progressing from an initial alteration in protein secretory dynamics (increased luminal MUCLIN and protein/mucus plugs) to an upregulation of glycoprotein/mucin gene expression, which is expected to exacerbate obstruction of the luminal spaces.

Upregulated Expression of Integrin α1 in Mesangial Cells and Integrin α3 and Vimentin in Podocytes of Col4a3-Null (Alport) Mice

Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen α3α4α5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ∼2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM.

Expression of sulfated gp300 and changes in glycosylation during pancreatic development.

The pancreatic zymogen granule membrane protein gp300 is the major sulfated glycoprotein of the mouse acinar cell and has been proposed to be an important structural component of the zymogen granule membrane. A prediction of this proposed function is that gp300 expression should be coordinately regulated with the digestive enzymes and appearance of zymogen granules during differentiation of acinar cells in fetal development. By Western blots and immunolocalization with a polyclonal antiserum to gp300, we found that gp300 protein expression paralleled expression of amylase and the appearance of zymogen granules in differentiating acinar cells. Lectin blots were performed to assess the glycoconjugate composition of gp300 during development. Using the fucose binding lectin Ulex europaeus I, we found that gp300 acquires this carbohydrate only postnatally, temporally correlated with weaning. In addition, gp300 showed complex changes during postnatal development in reactivity with the galactose binding lectin peanut agglutinin (PNA) and the sialic acid binding lectin Maackia amuresis (MAA). Levels of reactivity of PNA and MAA were reciprocal, suggesting that sialylation of galactose (which can block peanut agglutinin binding) was not constant on gp300 during development.

Transgenic Expression of Human LAMA5 Suppresses Murine Lama5 mRNA and Laminin α5 Protein Deposition

Laminin α5 is required for kidney glomerular basement membrane (GBM) assembly, and mice with targeted deletions of the Lama5 gene fail to form glomeruli. As a tool to begin to understand factors regulating the expression of the LAMA5 gene, we generated transgenic mice carrying the human LAMA5 locus in a bacterial artificial chromosome. These mice deposited human laminin α5 protein into basement membranes in heart, liver, spleen and kidney. Here, we characterized two lines of transgenics; Line 13 expressed ∼6 times more LAMA5 than Line 25. Mice from both lines were healthy, and kidney function and morphology were normal. Examination of developing glomeruli from fetal LAMA5 transgenics showed that the human transgene was expressed at the correct stage of glomerular development, and deposited into the nascent GBM simultaneously with mouse laminin α5. Expression of human LAMA5 did not affect the timing of the mouse laminin α1–α5 isoform switch, or that for mouse laminin β1–β2. Immunoelectron microscopy showed that human laminin α5 originated in both glomerular endothelial cells and podocytes, known to be origins for mouse laminin α5 normally. Notably, in neonatal transgenics expressing the highest levels of human LAMA5, there was a striking reduction of mouse laminin α5 protein in kidney basement membranes compared to wildtype, and significantly lower levels of mouse Lama5 mRNA. This suggests the presence in kidney of a laminin expression monitor, which may be important for regulating the overall production of basement membrane protein.