Functional and cellular characterization of human Retinoic Acid Induced 1 (RAI1) mutations associated with Smith-Magenis Syndrome

<p>Abstract</p> <p>Background</p> <p>Smith-Magenis Syndrome is a contiguous gene syndrome in which the dosage sensitive gene has been identified: the Retinoic Acid Induced 1 (<it>RAI1</it>). Little is known about the function of human RAI1.</p> <p>Results</p> <p>We generated the full-length cDNA of the wild type protein and five mutated forms: <it>RAI1-HA </it>2687delC, <it>RAI1-HA </it>3103delC, <it>RAI1 </it>R960X, <it>RAI1-HA </it>Q1562R, and <it>RAI1-HA </it>S1808N. Four of them have been previously associated with SMS clinical phenotype. Molecular weight, subcellular localization and transcription factor activity of the wild type and mutant forms were studied by western blot, immunofluorescence and luciferase assays respectively. The wild type protein and the two missense mutations presented a higher molecular weight than expected, localized to the nucleus and activated transcription of a reporter gene. The frameshift mutations generated a truncated polypeptide with transcription factor activity but abnormal subcellular localization, and the same was true for the 1-960aa N-terminal half of RAI1. Two different C-terminal halves of the RAI1 protein (1038aa-end and 1229aa-end) were able to localize into the nucleus but had no transactivation activity.</p> <p>Conclusion</p> <p>Our results indicate that transcription factor activity and subcellular localization signals reside in two separate domains of the protein and both are essential for the correct functionality of RAI1. The pathogenic outcome of some of the mutated forms can be explained by the dissociation of these two domains.</p

Podocyte-Specific Overexpression of Wild Type or Mutant Trpc6 in Mice Is Sufficient to Cause Glomerular Disease

Mutations in the TRPC6 calcium channel (Transient receptor potential channel 6) gene have been associated with familiar forms of Focal and Segmental Glomerulosclerosis (FSGS) affecting children and adults. In addition, acquired glomerular diseases are associated with increased expression levels of TRPC6. However, the exact role of TRPC6 in the pathogenesis of FSGS remains to be elucidated. In this work we describe the generation and phenotypic characterization of three different transgenic mouse lines with podocyte-specific overexpression of the wild type or any of two mutant forms of Trpc6 (P111Q and E896K) previously related to FSGS. Consistent with the human phenotype a non-nephrotic range of albuminuria was detectable in almost all transgenic lines. The histological analysis demonstrated that the transgenic mice developed a kidney disease similar to human FSGS. Differences of 2–3 folds in the presence of glomerular lesions were found between the non transgenic and transgenic mice expressing Trpc6 in its wild type or mutant forms specifically in podocytes. Electron microscopy of glomerulus from transgenic mice showed extensive podocyte foot process effacement. We conclude that overexpression of Trpc6 (wild type or mutated) in podocytes is sufficient to cause a kidney disease consistent with FSGS. Our results contribute to reinforce the central role of podocytes in the etiology of FSGS. These mice constitute an important new model in which to study future therapies and outcomes of this complex disease

Unveiling the biology of collecting duct epithelium repair and regeneration

The healthy functioning of the kidney requires the orchestrated action of its two functional units, nephrons and the collecting duct system (CD). Therefore malfunction of either of these two essential compartments can lead to kidney failure. Diseases affecting the collecting duct system (CD), congenital abnormalities of the kidney and urinary tract (CAKUTs), are the most frequent cause of End-Stage Renal Disease (ESRD) in children. At this stage, the only available therapeutic options for treating kidney failure are dialysis or organ transplantation. Given the fact that only one in three patients will receive a transplant, and that dialysis comes with a high risk of mortality, the study of the mechanisms underlying the repair and regeneration of the collecting duct system is vital because it will facilitate new therapeutic strategies for treating kidney disease which are available to more people. The CD system originates from one of the two progenitor populations which give rise to the kidney, the ureteric bud (UB). The formation of the UB is a substantial part of the nephrogenesis process, and develops into a branched tree-like structure which will ultimately form the ducts of the urinary collecting system. This PhD thesis investigates recent concepts in normal kidney organogenesis, repair, and regeneration, and focuses on the CD system. In the first part of this research, we investigated the capacity of an endogenous kidney mesenchymal stem cell population (k-MSC), previously reported by our laboratory, which both arose from the collecting duct epithelium and also then integrated into the same compartment. To this end, we generated and examined the functional capacity of Pkd1 defective k-MSCs to trigger autosomal dominant polycystic kidney disease (ADPKD) into wild type mice. Given that micro-injection of double transgenic k-MSCsPkd1del2-4/TMTO+ did not produce significant evidence for cyst formation in the recipient mice, we therefore ruled out the possibility that k-MSCs represent a preferable population for effecting repair. Nevertheless, we showed the capacity to generate k-MSCs with mutant genes, which may prove useful as cellular models of human diseases

Regenerative medicine in kidney disease

The treatment of renal failure has changed little in decades. Organ transplantation and dialysis continue to represent the only therapeutic options available. However, decades of fundamental research into the response of the kidney to acute injury and the processes driving progression to chronic kidney disease are beginning to open doors to new options. Similarly, continued investigations into the cellular and molecular basis of normal kidney development, together with major advances in stem cell biology, are now delivering options in regenerative medicine not possible as recently as a decade ago. In this review, we will discuss advances in regenerative medicine as it may be applied to the kidney. This will cover cellular therapies focused on ameliorating injury and improving repair as well as advancements in the generation of new renal tissue from stem/progenitor cells

Does Renal Repair Recapitulate Kidney Development?

Over a decade ago, it was proposed that the regulation of tubular repair in the kidney might involve the recapitulation of developmental pathways. Although the kidney cannot generate new nephrons after birth, suggesting a low level of regenerative competence, the tubular epithelial cells of the nephrons can proliferate to repair the damage after AKI. However, the debate continues over whether this repair involves a persistent progenitor population or any mature epithelial cell remaining after injury. Recent reports have highlighted the expression of Sox9, a transcription factor critical for normal kidney development, during postnatal epithelial repair in the kidney. Indeed, the proliferative response of the epithelium involves expression of several pathways previously described as being involved in kidney development. In some instances, these pathways are also apparently involved in the maladaptive responses observed after repeated injury. Whether development and repair in the kidney are the same processes or we are misinterpreting the similar expression of genes under different circumstances remains unknown. Here, we review the evidence for this link, concluding that such parallels in expression may more correctly represent the use of the same pathways in a distinct context, likely triggered by similar stressors

Characterization of a Trpc6 Transgenic Mouse Associated with Early Onset FSGS

Mutations in Transient Receptor Potential Channel 6 ( ) gene are associated with autosomal dominant focal and segmental glomerulosclerosis (FSGS). The majority of the identified mutations affect the ion channel function. Since calcium channels are promising candidate drug targets, there is an an urgent need for a mouse model to assess new therapeutic drugs and to help delineate the pathogenic process leading to FSGS. We have previously reported the generation of three independent transgenic mouse lines carrying different Trpc6 mutations that display a glomerular disease comparable to the phenotype presented by individuals with FSGS. However, the utility of these models for drug testing is dampened by the late-onset of the presentation and the mild phenotypic manifestations. In order to obtain a time-effective mouse model for Trpc6-associated FSGS we generated a new transgenic mutant Trpc6 mouse model emulating the amino acid change carried by the first pediatric patient of FSGS associated with a TRPC6 mutation: M132T. Mice carrying the orthologous Trpc6 M131T transgene showed early onset proteinuria and early signs of FSGS. When exploring molecular consequences of the overexpression of this mutated form of Trpc6 in podocytes, differences in expression levels of Axin2 and β-catenin were found in glomeruli from transgenic Trpc6 M131T mice. These data supports the proposed molecular mechanisms related to the activation of calcineurin-NFAT/Wnt signaling, as outcome of the increased calcium influx caused by the mutated form of Trpc6. Given that the Trpc6 M131T mouse develops an early onset of FSGS-like phenotypes it represents a promising model for studying the pathogenesis of FSGS caused by TRpC6, facilitating the assessment of new drugs as treatments and allowing further studies to understand underlying molecular pathways involved in the development of the TRPC6 mediated disease

Protein expression in transgenic mice.

<p>(A) The <i>in vivo</i> Trpc6-HA expression was confirmed by Immunoprecipitation (IP). The IP was performed from isolated mouse glomeruli (n = 8 kidney from each genotype) as described in material and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012859#s4" target="_blank">methods</a>. The glomeruli fraction was loaded into an HA affinity column, and a 30 µl aliquot of the sample which did not bind into the column was loaded in a gel and stained with Coomassie brilliant blue as a loading control (effluent control) for transgenic and non transgenic samples. (B) 30 µl of each IP sample was run in a SDS PAGE and a Western blot analysis against HA epitope was performed. (C) Double immunofluorescence of kidney cryosections to detect synaptopodin (green) and HA (red) for every transgenic line utilized in this study (400x).</p

Phenotypic characterization of kidney function in adult transgenic mice.

<p>Albuminuria levels (µg/dL) normalized by creatininuria levels (mg/dL) were tested in male mice at the age of 5–9 months. The number of mice analyzed for each genotype is as follows: non transgenic mice: n = 10, transgenic 419 n = 9, 421 n = 9, 615 n = 3, 616 n = 13, 73a n = 17, 75a n = 9. Data are presented in the bars as mean +/− SEM. * and ** mean statistical significance using non transgenic mice as control group with a <i>P</i> value <0.05 and <0.01, respectively.</p