Mi-2/NuRD is required in renal progenitor cells during embryonic kidney development.

Development of the nephron tubules, the functional units of the kidney, requires the differentiation of a renal progenitor population of mesenchymal cells to epithelial cells. This process requires an intricate balance between self-renewal and differentiation of the renal progenitor pool. Sall1 is a transcription factor necessary for renal development which is expressed in renal progenitor cells (cap mesenchyme). Sall1 recruits the Nucleosome Remodeling and Deacetylase (NuRD) chromatin remodeling complex to regulate gene transcription. We deleted Mi2ß, a component of the NuRD complex, in cap mesenchyme (CM) to examine its role in progenitor cells during kidney development. These mutants displayed significant renal hypoplasia with a marked reduction in nephrons. Markers of renal progenitor cells, Six2 and Cited1 were significantly depleted and progenitor cell proliferation was reduced. We also demonstrated that Sall1 and Mi2ß exhibited a strong in vivo genetic interaction in the developing kidney. Together these findings indicate that Sall1 and NuRD act cooperatively to maintain CM progenitor cells.

Exclusion of SIX6 hemizygosity in a child with anophthalmia, panhypopituitarism and renal failure.

We report a patient who presented with anophthalmia, panhypopituitarism, early onset of end stage renal failure, and craniofacial abnormalities. MRI at age 3 revealed that the pituitary was absent and renal biopsy demonstrated nephronophthisis as the cause of the renal failure. A similar syndrome has been associated with interstitial deletions of chromosome 14q22 and in one case hemizygosity for SIX6 was demonstrated. The patient reported here had a normal karyotype and Southern blot did not reveal loss of one copy of SIX6. We discuss other possible candidate genes that could be implicated in this syndrome.

The role of homeobox genes in kidney development.

Homeodomain-containing transcription factors are essential for a variety of processes in vertebrate development, including organogenesis. They have been shown to regulate cell proliferation, pattern segmental identity and determine cell fate decisions during embryogenesis. During nephrogenesis, homeobox genes play an important role at multiple developmental stages, from the early events in intermediate mesoderm to terminal differentiation of glomerular and tubular epithelia. Increasingly sophisticated genetic approaches will probably reveal additional functions for this class of transcription factors in the developing kidney and lead to the identification of critical downstream target genes.

BMP7 acts in murine lens placode development.

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation.

Induction of Rb-associated protein (RbAp46) by Wilms' tumor suppressor WT1 mediates growth inhibition.

The Wilms' tumor suppressor gene, wt1, encodes a zinc-finger transcription factor, WT1, that plays an important role in controlling urogenital development. Previously, WT1 has been shown to inhibit cell growth and to repress transcription initiated from the promoters of a number of growth-promoting genes. However, few physiological target genes that are transcriptionally activated by WT1 have been established. Using suppression subtractive hybridization polymerase chain reaction, we isolated a WT1 target gene that is up-regulated about 15-fold in cells expressing WT1. The gene was identified as retinoblastoma suppressor (Rb)-associated protein 46 (RbAp46), a nuclear protein that interacts physically with Rb and is a component of the human mSin3 co-repressor complex. Cells transfected with RbAp46 cDNA formed fewer colonies than the control cells, and RbAp46 suppressed the growth rate (by about 2-fold) of transfected cells. In the developing kidney and gonad, RbAp46 exhibits an expression pattern similar to that of WT1. We conclude that RbAp46 has strong growth inhibition activity and may function as an important mediator of WT1's function.

Lens-specific gene recruitment of zeta-crystallin through Pax6, Nrl-Maf, and brain suppressor sites.

Zeta-Crystallin is a taxon-specific crystallin, an enzyme which has undergone direct gene recruitment as a structural component of the guinea pig lens through a Pax6-dependent mechanism. Tissue specificity arises through a combination of effects involving three sites in the lens promoter. The Pax6 site (ZPE) itself shows specificity for an isoform of Pax6 preferentially expressed in lens cells. High-level expression of the promoter requires a second site, identical to an alphaCE2 site or half Maf response element (MARE), adjacent to the Pax6 site. A promoter fragment containing Pax6 and MARE sites gives lens-preferred induction of a heterologous promoter. Complexes binding the MARE in lens nuclear extracts are antigenically related to Nrl, and cotransfection with Nrl elevates zeta-crystallin promoter activity in lens cells. A truncated zeta promoter containing Nrl-MARE and Pax6 sites has a high level of expression in lens cells in transgenic mice but is also active in the brain. Suppression of the promoter in the brain requires sequences between -498 and -385, and a site in this region forms specific complexes in brain extract. A three-level model for lens-specific Pax6-dependent expression and gene recruitment is suggested: (i) binding of a specific isoform of Pax6; (ii) augmentation of expression through binding of Nrl or a related factor; and (iii) suppression of promoter activity in the central nervous system by an upstream negative element in the brain but not in the lens.

Induction of molecular chaperones by hyperosmotic stress in mouse inner medullary collecting duct cells.

The extreme hyperosmotic conditions that exist in the renal inner medulla enable the urinary concentrating mechanism to function. In this study, we evaluated whether stress-related molecular chaperones are induced in response to hyperosmotic stress in mouse inner medullary collecting duct (mIMCD3) cells. Exposure of cells to medium supplemented with 100 mM NaCl for 4 or 24 h resulted in an increase in heat shock protein-72 (HSP-72) (inducible form) by Western blot. Immunocytochemistry confirmed the increase of HSP-72 and showed that hyperosmotic stress resulted in a localization of HSP-72 predominantly to the nucleoplasm that surrounds the nucleoli and to the cytoplasm, a subcellular distribution pattern different from that seen with heat shock. Using a denatured protein (casein)-affinity column with ATP elution, we identified a number of putative molecular chaperones (46, 60, 78, and 200 kDa) that are upregulated in response to 4 h of hyperosmotic NaCl treatment. Microsequencing identified one of these proteins to be the mitochondrial chaperone mtHSP-70, a member of HSP-70 family, and another to be similar to beta-actin. We also found high levels of HSP-72 in cells chronically adapted to hypertonicity, indicating that chaperones are still required to maintain certain cellular functions even after nonperturbing organic osmolytes are known to accumulate. These results suggest an important role for molecular chaperones in the adaptation of renal medullary epithelial cells to the hyperosmotic conditions that exist in the inner medulla in vivo.

Molecular cloning and chromosome localization of a putative basolateral Na(+)-K(+)-2Cl- cotransporter from mouse inner medullary collecting duct (mIMCD-3) cells.

Electroneutral Na(+)-K(+)-2Cl- cotransporters represent one of the major routes for Cl- movement in epithelia. A secretory form of the cotransporter has been described in the basolateral membrane of a variety of epithelia from fish to mammals. We isolated a putative bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter cDNA, BSC2, from mIMCD-3 cells. Northern analysis indicates that in contrast to BSC1, the recently cloned renal-specific apical isoform of the cotransporter, BSC2 is expressed in secretory epithelia and thus appears to represent the basolateral isoform. Furthermore, BSC2 is also expressed in non-polarized cells, such as red cells and myocytes. Sequence comparison and chromosome localization demonstrate that BSC2 and BSC1 are different genes that diverged before the evolution of vertebrates.

Regulation of mitogenesis, motogenesis, and tubulogenesis by hepatocyte growth factor in renal collecting duct cells.

Hepatocyte growth factor (HGF) has been implicated in branching tubulogenesis of the developing kidney and in response to renal injury. We therefore examined the effects of response to renal injury. We therefore examined the effects of HGF on a recently described murine inner medullary collecting duct epithelial cell line (mIMCD-3 cells) in comparison with Madin-Darby canine kidney (MDCK) cells. HGF induced mitosis, scattering, and tubulogenesis in both mIMCD-3 cells and MDCK cells. However, mIMCD-3 cells underwent branching tubulogenesis under matrix conditions that did not support these morphogenetic changes in MDCK cells, suggesting substantial differences in regulation of tubulogenesis in these two cell types. In quiescent mIMCD-3 cells, the high-affinity receptor for HGF, c-met, was expressed in a nonphosphorylated state. After stimulation with HGF, there was a > 10-fold increase in receptor tyrosine phosphorylation and selective association with at least two intracellular proteins, including the phosphatidylinositol-3-kinase. Thus mIMCD-3 cells, which undergo HGF-dependent mitosis, scattering, and branching tubulogenesis, express the c-met receptor in a highly regulated state and therefore should make an excellent model for examining the mechanisms of HGF-dependent tubulogenesis in the renal collecting duct.

An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse.

The terminal inner medullary collecting duct (IMCD) plays an important role in determining the final urinary composition. Currently, there is no continuous cell line derived from this nephron segment. We have developed a cell line derived from the terminal IMCD of mice transgenic for the early region of simian virus SV40 (large T antigen). This cell line, mIMCD-3, retains many differentiated characteristics of this nephron segment including high transepithelial resistance (1,368 +/- 172 omega.cm2), inhibition of apical-to-basal sodium flux by amiloride (41 +/- 7%) and by atrial natriuretic peptide (ANP) (40 +/- 9%), the presence of the amiloride-sensitive sodium channel as determined by Western blot analysis, and accumulation of the major organic osmolytes in response to hypertonic stress. Significantly, mIMCD-3 cells adapted readily and were able to grow in hypertonic medium supplemented with NaCl and urea up to 910 mosmol/kgH2O. These extreme osmotic conditions exist in the renal medulla in vivo but are known to be lethal to most other cells. This cell line should be highly useful for the study of the cellular adaptation to osmotic stress and the cell biology and transport physiology of this nephron segment.

Expression of GLUT-2 cDNA in human B lymphocytes: analysis of glucose transport using flow cytometry.

The molecular characterization of transport proteins is often limited by transient functional expression or the need for a simple method to select functional cDNA clones. We used a mammalian expression system to obtain long-term expression of GLUT-2, an isoform of glucose permease. Rat GLUT-2 cDNA was ligated into an EBV vector (pLPP) and transfected into B lymphocytes which lack GLUT-2. Northern and Western analyses confirmed expression of GLUT-2 protein in membranes of transfected cells. Two functional assays using flow cytometry were developed to distinguish GLUT-2 transfectants from control/pLPP transfectants. Uptake of NBD-glucosamine, a fluorescent analogue of glucose, was increased in GLUT-2 transfectants. In addition, when exposed to hypertonic glucose medium, GLUT-2 transfectants and control/pLPP transfectants exhibited a difference in forward-angle light scatter (FALS), an index of cell volume, indicating a difference in glucose permeability. Independent measurements of glucose uptake (isotopic) and cell volume (video microscopy) confirmed the flow cytometry observations. This expression system used in combination with flow cytometry is useful for studying the functional properties of glucose and other solute transporters.