Polycystic Kidney Disease in the Medaka (Oryzias latipes) pc Mutant Caused by a Mutation in the Gli-Similar3 (glis3) Gene

Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients

The effect of rapamycin on biodiesel-producing protist <i>Euglena gracilis</i>

<p>Rapamycin induces autophagy with lipid remodeling in yeast and mammalian cells. To investigate the lipid biosynthesis of <i>Euglena gracilis</i>, rapamycin was supplemented in comparison with two model algae, <i>Chlamydomonas reinhardtii</i> and <i>Cyanidioschyzon merolae</i>. In <i>Euglena</i>, rapamycin induced the reduction of chlorophylls and the accumulation of neutral lipids without deterring its cell proliferation. Its lipidomic profile revealed that the fatty acid composition did not alter by supplementing rapamycin. In <i>Chlamydomonas</i>, however, rapamycin induced serious growth inhibition as reported elsewhere. With a lower concentration of rapamycin, the alga accumulated neutral lipids without reducing chlorophylls. In <i>Cyanidioschyzon</i>, rapamycin did not increase neutral lipids but reduced its chlorophyll content. We also tested fatty acid elongase inhibitors such as pyroxasulfone or flufenacet in <i>Euglena</i> with no significant change in its neutral lipid contents. In summary, controlled supplementation of rapamycin can increase the yield of neutral lipids while the scheme is not always applicable for other algal species.</p> <p>Lipid profile of <i>Euglena gracilis</i> under supplementation of rapamycin. With <10 µM rapamycin, the neutral lipid contents increase without deterring cell growth.</p

Monocytes Infiltrate the Pancreas via the MCP-1/CCR2 Pathway and Differentiate into Stellate Cells

<div><p>Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP)⁺CD45^– cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl₄). Because the vast majority of EGFP⁺CD45^– cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs). EGFP⁺ PaSCs were also observed in CCl₄-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) increased in the pancreas of CCl₄-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2) and Ang II type 1 receptor (AT1R), were expressed on Ly6C^high monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II <i>in vitro</i>. Irbesartan inhibited not only their <i>in vitro</i> chemotaxis but also <i>in vivo</i> migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP⁺F4/80⁺CCR2⁺ monocytic cells and EGFP⁺ PaSCs in the pancreas of CCl₄-treated chimeric mice receiving EGFP⁺ bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP⁺ PaSCs in injured mice. We propose that CCR2⁺ monocytes migrate into the pancreas possibly via the MCP-1/CCR2 pathway and give rise to PaSCs.</p></div

Experimental design.

<p>(A) Clones from a CD34^–c-kit⁺Sca-1⁺lineage^– (CD34^–KSL) cell or a total of 2×10⁶ bone marrow-total nucleated cells (BM-TNCs) isolated from enhanced green fluorescent protein (EGFP)-transgenic mice were transplanted into lethally irradiated C57BL/6J-Ly5.1 mice. Two months after BM transplantation, mice were intraperitoneally injected with CCl₄ or olive oil twice a week for 12 weeks. (B) C57BL/6J-Ly5.1 mice were intraperitoneally injected with CCl₄ twice weekly for 5 weeks. Ly6C⁺ monocytes, peripheral blood (PB)-TNCs, or a PB-Ly6C⁺ cell-depleted population isolated from EGFP-transgenic mice were adoptively transferred into CCl₄-treated mice at 24 hours after each injection of CCl₄ for 2 weeks. (C) C57BL/6J-Ly5.1 mice were intraperitoneally injected with CCl₄ twice weekly for 5 weeks. They were fed chow containing irbesartan or normal chow for 2 weeks. Ly6C⁺ monocytes isolated from EGFP-transgenic mice were adoptively transferred into CCl₄-treated mice at 24 hours after each injection of CCl₄ for 2 weeks. (D) C57BL/6J-Ly5.1 mice that received 2×10⁶ EGFP⁺ BM-TNCs were fed chow containing irbesartan or normal chow for 6 weeks. In another group of mice, RS504393 or vehicle was subcutaneously administered once a day for 6 weeks. One week after the initiation of irbesartan or RS504393 treatment, CCl₄ treatment was started and continued for 5 weeks.</p

Hematopoietic engraftment in mice transplanted with clonal cells derived from a single CD34^–KSL cell.

<p>EGFP, enhanced green fluorescent protein; CD34^–KSL cell,</p><p>CD34^–c-kit⁺Sca-1⁺lienage^– cell.</p

MCP-1 and angiotensinogen production in pancreas of CCl₄-treated mice and their receptor expression on monocytes.

<p>(A) The number of engrafted EGFP⁺ cells and the percentages of CD45^–GFAP⁺ cells among EGFP⁺ cells in the pancreases of mice that received Ly6C^highc-kit^– cells, PB-TNCs, or a PB-Ly6C⁺ cell-depleted population isolated from EGFP-transgenic mice are shown. Data are the means ± SD of three mice. (B) Total RNA isolated from the pancreases from four untreated mice, four olive oil-treated mice, and three CCl₄-treated mice was analyzed by RT-PCR for mRNA expression of MCP-1 and angiotensinogen. The level of mRNA was normalized to that of GAPDH mRNA. Data are the means ± SD of three or four mice per group. *<i>P</i><0.05 versus untreated mice or olive oil-treated mice. (C) Total RNA from Ly6C⁺ monocytes, which were isolated from the BM of naive EGFP mice, was analyzed by RT-PCR for mRNA expression of CCR2, AT1Ra, and AT1Rb. Representative examples of CCR2, AT1Ra, and AT1Rb mRNA expression are shown in the left panel. The expression of CCR2 and AT1R on Ly6C⁺ monocytes was evaluated by single-color flow cytometry. Representative examples of histograms for CCR2 and AT1R expression are shown in the center and right panel, respectively. Black lines indicate isotype control staining.</p