Uninephrectomy and apical fluid shear stress decrease ENaC abundance in collecting duct principal cells.

Acute nephron reduction such as after living kidney donation may increase the risk of hypertension. Uninephrectomy induces major hemodynamic changes in the remaining kidney, resulting in rapid increase of single-nephron glomerular filtration rate (GFR) and fluid delivery in the distal nephron. Decreased sodium (Na) fractional reabsorption after the distal tubule has been reported after uninephrectomy in animals preserving volume homeostasis. In the present study, we thought to specifically explore the effect of unilateral nephrectomy on epithelial Na channel (ENaC) subunit expression in mice. We show that γ-ENaC subunit surface expression was specifically downregulated after uninephrectomy, whereas the expression of the aldosterone-sensitive α-ENaC and α1-Na-K-ATPase subunits as well as of kidney-specific Na-K-Cl cotransporter isoform and Na-Cl cotransporter were not significantly altered. Because acute nephron reduction induces a rapid increase of single-nephron GFR, resulting in a higher tubular fluid flow, we speculated that local mechanical factors such as fluid shear stress (FSS) were involved in Na reabsorption regulation after uninephrectomy. We further explore such hypothesis in an in vitro model of FSS applied on highly differentiated collecting duct principal cells. We found that FSS specifically downregulates ß-ENaC and γ-ENaC subunits at the transcriptional level through an unidentified heat-insensitive paracrine basolateral factor. The primary cilium as a potential mechanosensor was not required. In contrast, protein kinase A and calcium-sensitive cytosolic phospholipase A2 were involved, but we could not demonstrate a role for cyclooxygenase or epoxygenase metabolites.

R-spondin1 deficiency in mice improves glycaemic control in association with increased beta cell mass.

AIMS/HYPOTHESIS: Roof plate-specific spondin (R-spondin1; RSPO1) is a modulator of canonical Wg (wingless) plus Int1 (chromosomal integration site of mouse mammary tumour virus on mouse chromosome 15) (cWNT) signalling that induces cWNT target genes. We have demonstrated that Rspo1 is expressed in murine beta cells, and that it stimulates proliferation and insulin secretion, and inhibits cytokine-induced apoptosis, in mouse insulinoma (MIN6) and beta cells. We thus investigated the role of RSPO1 in beta cells in vivo using Rspo1 ( -/- ) mice. METHODS: The effects of Rspo1 deficiency were assessed by determination of cWNT signalling, glucose tolerance and beta cell mass. RESULTS: Rspo1 ( -/- ) mice demonstrated an 82% reduction in RSPO1 transcripts and a 61% reduction in the signal detected by an RSPO1 antibody, as well as a 47% decrease in islet cWNT signalling. Despite no differences in body and pancreatic weights or in fasting glycaemia and insulinaemia compared with Rspo1 (+/+) mice, Rspo1 ( -/- ) animals had improved glycaemic control after oral glucose challenge (p < 0.05), with no difference in insulin sensitivity, but an enhanced insulin response over 30 min (p < 0.05); glucagon responses were normal. Rspo1 deficiency also resulted in a twofold increase in beta cell mass (p < 0.05) in association with 2- and 12-fold increases in the number of beta cells positive for antigen identified by monoclonal antibody Ki67 (Ki67) (p < 0.01) and insulin-positive ductal cells (p < 0.05), respectively. No change in the number of TUNEL-positive beta cells was detected. Islets isolated from Rspo1 ( -/- ) animals displayed no differences in glucose-induced insulin secretion or in glucose suppression of glucagon. CONCLUSIONS/INTERPRETATION: The present study reveals an unexpected role for RSPO1 as a regulator of both beta cell proliferation and neogenesis in vivo, and reinforces the importance of cWNT signalling for the maintenance of normal pancreatic beta cell behaviour.

Id3 is a novel regulator of p27kip1 mRNA in early G1 phase and is required for cell-cycle progression.

P27kip is a key inhibitory protein of the cell-cycle progression, which is rapidly downregulated in early G1 phase by a post-translational mechanism involving the proteosomal degradation. In this study, using a wounding model that induces cell-cycle entry of human dermal fibroblasts, we demonstrate that p27mRNA is downregulated when cells progress into the G1 phase, and then it returns to its basal level when cells approach the S phase. By using a quantitative polymerase chain reaction screening we identified inhibitors of differentiation (Id3), a bHLH transcriptional repressor, as a candidate mediator accounting for p27 mRNA decrease. Id3 silencing, using an small interfering RNA approach, reversed the injury mediated p27 downregulation demonstrating that Id3 is involved in the transcriptional repression of p27. Reporter gene experiments and a chromatin immunoprecipitation assay showed that Id3 likely exerts its repressive action through ELK1 inhibition. By inhibiting early p27 downregulation, Id3 depletion blocked (i) the G1-phase progression as assessed by the inhibition of pRb phosphorylation and p130 degradation and (ii) the G1/S transition as observed by the inhibition of cyclin A induction, demonstrating that p27 mRNA decrease is required for cell proliferation. Apart from its effect on the early p27 diminution, Id3 appears also involved in the control of the steady-state level of p27 at the G1/S boundary. In conclusion, this study identifies a novel mechanism of p27 regulation which besides p27 protein degradation also implicates a transcriptional mechanism mediated by Id3.

Amplification of R-spondin1 signaling induces granulosa cell fate defects and cancers in mouse adult ovary.

R-spondin1 is a secreted regulator of WNT signaling, involved in both embryonic development and homeostasis of adult organs. It can have a dual role, acting either as a mitogen or as a tumor suppressor. During ovarian development, Rspo1 is a key factor required for sex determination and differentiation of the follicular cell progenitors, but is downregulated after birth. In human, increased RSPO1 expression is associated with ovarian carcinomas, but it is not clear whether it is a cause or a consequence of the tumorigenic process. To address the role of Rspo1 expression in adult ovaries, we generated an Rspo1 gain-of-function mouse model. Females were hypofertile and exhibited various ovarian defects, ranging from cysts to ovarian tumors. Detailed phenotypical characterization showed anomalies in the ovulation process. Although follicles responded to initial follicle-stimulating hormone stimulation and developed normally until the pre-ovulatory stage, they did not progress any further. Although non-ovulated oocytes degenerated, the surrounding follicular cells did not begin atresia. RSPO1-induced expression not only promotes canonical WNT signaling but also alters granulosa cell fate decisions by maintaining epithelial-like traits in these cells. This prevents follicle cells from undergoing apoptosis, leading to the accumulation of granulosa cell tumors that reactivates the epithelial program from their progenitors. Taken together, our data demonstrate that activation of RSPO1 is sufficient in promoting ovarian tumors and thus supports a direct involvement of this gene in the commencement of ovarian cancers.

Loss of R-spondin1 and Foxl2 amplifies female-to-male sex reversal in XX mice.

In vertebrates, 2 main genetic pathways have been shown to regulate ovarian development. Indeed, a loss of function mutations in Rspo1 and Foxl2 promote partial female-to-male sex reversal. In mice, it has been shown that the secreted protein RSPO1 is involved in ovarian differentiation and the transcription factor FOXL2 is required for follicular formation. Here, we analysed the potential interactions between these 2 genetic pathways and have shown that while Rspo1 expression seems to be independent of Foxl2 up-regulation, Foxl2 expression partly depends of Rspo1 signalisation. This suggests that different Foxl2-positive somatic cell lineages exist within the ovaries. In addition, a combination of both mutated genes in XX Foxl2(-/-)/Rspo1(-/-) gonads promotes sex reversal, detectable at earlier stages than in XX Rspo1(-/-) mutants. Ectopic development of the steroidogenic lineage is more pronounced in XX Foxl2(-/-)/Rspo1(-/-) gonads than in XX Rspo1(-/-) embryos, suggesting that Foxl2 is involved in preventing ectopic steroidogenesis in foetal ovaries.

Genetics of ovarian differentiation: Rspo1, a major player.

In mammals, the sex of the embryo is determined during development by its commitment either to the male or female genetic program regulating testicular or ovarian organogenesis. Major steps towards unraveling sex determination in mammals are achieved by the identification of key genes involved in human pathologies and the application of mouse genetics to analyze their function. While the expression of Sry and Sox9 is sufficient to induce the male developmental program, the molecular pathways that specify ovarian differentiation were unclear before the recent demonstration that mutations in the RSPO1 gene induce female-to-male sex reversal in XX patients. By generating the corresponding mouse model, we have shown that Rspo1 is so far the earliest known gene controlling the female genetic developmental program. Rspo1 activates the canonical beta-catenin signaling pathway required for female somatic cell differentiation and germ cell commitment into meiosis. The aim of this review is to describe the roles of R-spondins (Rspo)in developmental processes and disorders and the current knowledge obtained from murine models. A particular focus will be on Rspo1 and its crucial function in sex determination.

Canal shapes produced sequentially during instrumentation with Quantec SC rotary nickel-titanium instruments: a study in simulated canals.

AIM: The aim of this study was to determine the shaping ability of Quantec SC nickel-titanium rotary instruments in simulated root canals. METHODOLOGY: Forty simulated canals consisting of four different shapes in terms of angle and position of curvature were prepared with Quantec SC instruments. Sequential still images were taken of the canals using a video camera attached to a computer with image analysis software. Images were taken preoperatively, and then after instrument 7 (Size 25, 0.05 taper), instrument 8 (size 25, 0.06 taper), and instrument 10 (size 45, 0.02 taper) were taken to length. Each sequential postoperative image was superimposed individually over the preoperative image in order to highlight the amount and position of material removed during preparation. RESULTS: Overall, the mean preparation time to size 10 was 3.6 min with 12 mm canals taking on average less time than 8 mm canals. There was a highly significant difference between the canal types (P < 0.0001). No instruments fractured within the canal or deformed, although one instrument separated from the latch grip. All canals remained patent. Following preparation to size 10, 19 canals (48%) retained their length, eight (20%) lost length, and 13 (32%) gained length; the magnitude of the change in length was always 0.5 mm or below. Following preparation to size 7 instruments all canals showed aberrant shapes. Excess removal of material along the outer aspect of the curve between the beginning of the curve and the end-point (outer widening) was found in 26 canals (65%) after instrument 7. At the same stage of preparation six canals (15%) had zips, three (8%) had ledges and five (13%) had perforations. Following preparation to size 10, 27 (68%) canals were perforated. CONCLUSIONS: Under the conditions of the study, Quantec SC instruments consistently produced aberrations when canals were enlarged to size 7 (size 25, 0.05 taper) or above. Care should be exercised when using these instruments in real teeth.