Marie Trudel

Overexpression of PKD1 Causes Polycystic Kidney Disease

ABSTRACT The pathogenetic mechanisms underlying autosomal dominant polycystic kidney disease (ADPKD) remain to be elucidated. While there is evidence that Pkd1 gene haploinsufficiency and loss of heterozygosity can cause cyst formation in mice, paradoxically high levels of Pkd1 expression have been detected in the kidneys of ADPKD patients. To determine whether Pkd1 gain of function can be a pathogenetic process, a Pkd1 bacterial artificial chromosome (Pkd1-BAC) was modified by homologous recombination to solely target a sustained Pkd1 expression preferentially to the adult kidney. Several transgenic lines were generated that specifically overexpressed the Pkd1 transgene in the kidneys 2- to 15-fold over Pkd1 endogenous levels. All transgenic mice reproducibly developed tubular and glomerular cysts and renal insufficiency and died of renal failure. This model demonstrates that overexpression of wild-type Pkd1 alone is sufficient to trigger cystogenesis resembling human ADPKD. Our results also uncovered a striking increased renal c-myc expression in mice from all transgenic lines, indicating that c-myc is a critical in vivo downstream effector of Pkd1 molecular pathways. This study not only produced an invaluable and first PKD model to evaluate molecular pathogenesis and therapies but also provides evidence that gain of function could be a pathogenetic mechanism in ADPKD.

Treatment with oral clotrimazole blocks Ca(2+)-activated K+ transport and reverses erythrocyte dehydration in transgenic SAD mice. A model for therapy of sickle cell disease.

Prevention of red cell K+ and water loss is a therapeutic strategy for sickle cell disease. We have investigated in vitro and in vivo the effects of clotrimazole (CLT) and miconazole (MIC) on transgenic mice red cells expressing hemoglobin SAD. CLT blocked the Gardos channel (ID50 75 +/- 22 nM; n = 3) and the A23187-induced dehydration of Hbbs/Hbbthal SAD 1 mouse erythrocytes in vitro. Oral treatment with CLT (160 mg/kg per d) and MIC (100 mg/kg per d) inhibited the Gardos channel in both SAD 1 and control (Hbbs/Hbbthal) mice. In the SAD 1 mice only, cell K+ content increased, and mean corpuscular hemoglobin concentration and cell density decreased. After 7 d of treatment, the hematocrit of SAD 1, CLT-treated animals also increased. All changes were fully reversible. Long-term treatments of SAD 1 mice with oral CLT (80 mg/kg per d for 28 d) lead to sustained increases in cell K+ content and hematocrit and sustained decreases in mean corpuscular hemoglobin concentration and cell density, with no changes in animals treated with vehicle alone. Thus, CLT and MIC can reverse dehydration and K+ loss of SAD 1 mouse erythrocytes in vitro and in vivo, further supporting the potential utility of these drugs in the treatment of sickle cell anemia.

Disruption of erythroid K-Cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice

K-Cl cotransport activity in rbc is a major determinant of rbc volume and density. Pathologic activation of erythroid K-Cl cotransport activity in sickle cell disease contributes to rbc dehydration and cell sickling. To address the roles of individual K-Cl cotransporter isoforms in rbc volume homeostasis, we disrupted the Kcc1 and Kcc3 genes in mice. As rbc K-Cl cotransport activity was undiminished in Kcc1(-/-) mice, decreased in Kcc3(-/-) mice, and almost completely abolished in mice lacking both isoforms, we conclude that K-Cl cotransport activity of mouse rbc is mediated largely by KCC3. Whereas rbc of either Kcc1(-/-) or Kcc3(-/-) mice were of normal density, rbc of Kcc1(-/-)Kcc3(-/-) mice exhibited defective volume regulation, including increased mean corpuscular volume, decreased density, and increased susceptibility to osmotic lysis. K-Cl cotransport activity was increased in rbc of SAD mice, which are transgenic for a hypersickling human hemoglobin S variant. Kcc1(-/-)Kcc3(-/-) SAD rbc lacked nearly all K-Cl cotransport activity and exhibited normalized values of mean corpuscular volume, corpuscular hemoglobin concentration mean, and K(+) content. Although disruption of K-Cl cotransport rescued the dehydration phenotype of most SAD rbc, the proportion of the densest red blood cell population remained unaffected.

Ikaros and GATA-1 Combinatorial Effect Is Required for Silencing of Human γ-Globin Genes

ABSTRACT During development and erythropoiesis, globin gene expression is finely modulated through an important network of transcription factors and chromatin modifying activities. In this report we provide in vivo evidence that endogenous Ikaros is recruited to the human β-globin locus and targets the histone deacetylase HDAC1 and the chromatin remodeling protein Mi-2 to the human γ-gene promoters, thereby contributing to γ-globin gene silencing at the time of the γ- to β-globin gene transcriptional switch. We show for the first time that Ikaros interacts with GATA-1 and enhances the binding of the latter to different regulatory regions across the locus. Consistent with these results, we show that the combinatorial effect of Ikaros and GATA-1 impairs close proximity between the locus control region and the human γ-globin genes. Since the absence of Ikaros also affects GATA-1 recruitment to GATA-2 promoter, we propose that the combinatorial effect of Ikaros and GATA-1 is not restricted to globin gene regulation.

Genetic correction of sickle cell disease: insights using transgenic mouse models.

Sickle cell disease is a hereditary disorder characterized by erythrocyte deformity due to hemoglobin polymerization. We assessed in vivo the potential curative threshold of fetal hemoglobin in the SAD transgenic mouse model of sickle cell disease using mating with mice expressing the human fetal Aγ-globin gene. With increasing levels of HbF, AγSAD mice showed considerable improvement in all hematologic parameters, morphopathologic features and life span/survival. We established the direct therapeutic effect of fetal hemoglobin on sickle cell disease and demonstrated correction by increasing fetal hemoglobin to about 9–16% in this mouse model. This in vivo study emphasizes the potential of the SAD mouse models for quantitative analysis of gene therapy approaches.

Murine Pkd1 is a developmentally regulated gene from morula to adulthood: role in tissue condensation and patterning.

PKD1 is the most common genetically mutated gene involved in autosomal dominant polycystic kidney disease (ADPKD). Our previous studies have shown that the pathogenesis of human and murine polycystic kidney disease (PKD) involves failure to switch out of a renal developmental program, suggesting a role for PKD1 in development. To investigate this hypothesis, we have cloned a portion of the murine Pkd1 gene and characterized the fetal to adult tissue expression pattern of Pkd1. We chose to clone the transmembrane region of Pkd1, a region prone to mutations in ADPKD. The transmembrane coding region (2.6 kb) has 80.3% nucleotide homology with human PKD1 and 85.3% amino acid similarity. The cloned murine Pkd1 fragment closely resembles that of human PKD1 with respect to both genomic size and exon/intron position. We have demonstrated that this Pkd1 region is not conserved in lower organisms and is mammalian specific. A detailed expression analysis of Pkd1 revealed expression as early as the morula stage and in ES cells with differential expression levels in various tissues/organs throughout development. Highest expression levels were observed in the early condensing mesenchyme of primitive mesoderm and ectoderm. Pkd1 was also expressed at high levels in developing neural tube, neural crest derivatives, prechondrogenic tissue, metanephros, bladder, salivary glands, lung, and blood vessels with lower expression levels in other organs and tissues. Specific spatial and temporal patterns of Pkd1 expression were demonstrated in individual organs, such as lung, kidney, brain, indicating it is highly developmentally regulated. Particularly high levels persisted in mature derivatives of neural tube, neural crest, chondrogenic tissue, metanephros, and lung. In summary, our data suggest that Pkd1 has at least two cellular functions, one a basic function involved in early tissue condensation processes, and the other a mammalian‐specific function, that evolved with tissue patterning and tubulogenesis in metanephric and pulmonary development. Dev Dyn 1999;214:337–348.

Pkd1 transgenic mice: adult model of polycystic kidney disease with extrarenal and renal phenotypes

While high levels of Pkd1 expression are detected in tissues of patients with autosomal dominant polycystic kidney disease (ADPKD), it is unclear whether enhanced expression could be a pathogenetic mechanism for this systemic disorder. Three transgenic mouse lines were generated from a Pkd1-BAC modified by introducing a silent tag via homologous recombination to target a sustained wild-type genomic Pkd1 expression within the native tissue and temporal regulation. These mice specifically overexpressed the Pkd1 transgene in extrarenal and renal tissues from approximately 2- to 15-fold over Pkd1 endogenous levels in a copy-dependent manner. All transgenic mice reproducibly developed tubular and glomerular cysts leading to renal insufficiency. Interestingly, Pkd1(TAG) mice also exhibited renal fibrosis and calcium deposits in papilla reminiscent of nephrolithiasis as frequently observed in ADPKD. Similar to human ADPKD, these mice consistently displayed hepatic fibrosis and approximately 15% intrahepatic cysts of the bile ducts affecting females preferentially. Moreover, a significant proportion of mice developed cardiac anomalies with severe left-ventricular hypertrophy, marked aortic arch distention and/or valvular stenosis and calcification that had profound functional impact. Of significance, Pkd1(TAG) mice displayed occasional cerebral lesions with evidence of ruptured and unruptured cerebral aneurysms. This Pkd1(TAG) mouse model demonstrates that overexpression of wild-type Pkd1 can trigger the typical adult renal and extrarenal phenotypes resembling human ADPKD.

C-myc as a modulator of renal stem/progenitor cell population

The role of c‐myc has been well‐studied in gene regulation and oncogenesis but remains elusive in murine development from midgestation. We determined c‐myc function during kidney development, organogenesis, and homeostasis by conditional loss of c‐myc induced at two distinct phases of nephrogenesis, embryonic day (e) 11.5 and e17.5. Deletion of c‐myc in early metanephric mesenchyme (e11.5) led to renal hypoplasia from e15.5 to e17.5 that was sustained until adulthood (range, 20–25%) and, hence, reproduced the human pathologic condition of renal hypoplasia. This phenotype resulted from depletion of c‐myc–positive cells in cap mesenchyme, causing a ∼35% marked decrease of Six2‐ and Cited1‐stem/progenitor population and of proliferation that likely impaired self‐renewal. By contrast, c‐myc loss from e17.5 onward had no impact on late renal differentiation/maturation and/or homeostasis, providing evidence that c‐myc is dispensable during these phases. This study identified c‐myc as a modulator of renal organogenesis through regulation of stem/progenitor cell population. Developmental Dynamics 238:405–414, 2009.

Hypoxia Activates a Ca2+-Permeable Cation Conductance Sensitive to Carbon Monoxide and to GsMTx-4 in Human and Mouse Sickle Erythrocytes

Background Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca2+] ([Ca2+]i) and subsequent activation of KCa 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration. Methodology/Principal Findings We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca2+- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 µM), dipyridamole (100 µM), DIDS (100 µM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca2+]i, in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca2+]i in mouse sickle erythrocytes did not require KCa3.1 activity. Conclusions/Significance The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca2+-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca2+ for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

Distinct and common developmental expression patterns of the murine Pkd2 and Pkd1 genes

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most commonly inherited renal diseases. At least two genes, PKD2 and PKD1 are implicated in the development of this disease. Our pathogenetic studies showed that the human and murine polycystic kidney disease (PKD) involves failure to switch out of a renal developmental program. We have thus undertaken a detailed comparative expression analysis of Pkd2 and Pkd1 from the morula stage to adulthood. Pkd2 expression was detected as early as the morula and blastocyst stages as observed for Pkd1. Strong Pkd2 expression, similar to Pkd1, was displayed in all mesenchymal and cartilaginous tissues during mouse development. However major differences in Pkd2 expression in comparison to Pkd1 were identified. First, in contrast to Pkd1, the neural crest cell-derived tissues displayed a low to undetectable Pkd2 expression at all ages. Second, no increase in Pkd2 expression was detected during mesenchymal condensation. Third, high Pkd2 expression in the kidneys was localized mainly to the tubular epithelium of the cortical region from murine development to adulthood.