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Background Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called “pronuclear injection-based targeted transgenesis” (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT ( i -PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre -mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre- LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. Results To enable the i -PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. Conclusions Simultaneous use of PhiC31 and FLP in i -PITT approach allows insertion of donor plasmids containing Cre- loxP -based conditional expression cassettes.

Foxc2 is a single-exon gene and a key regulator in development of multiple organs, including kidney. To avoid embryonic lethality of conventional Foxc2 knockout mice, we conditionally deleted Foxc2 in kidneys. Conditional targeting of a single-exon gene involves the large floxed gene segment spanning from promoter region to coding region to avoid functional disruption of the gene by the insertion of a loxP site. Therefore, in ES cell clones surviving a conventional single-selection, e.g., neomycin-resistant gene (neo) alone, homologous recombination between the long floxed segment and target genome results in a high incidence of having only one loxP site adjacent to the selection marker. To avoid this limitation, we employed a double-selection system. We generated a Foxc2 targeting construct in which a floxed segment contained 4.6 kb mouse genome and two different selection marker genes, zeocin-resistant gene and neo, that were placed adjacent to each loxP site. After double-selection by zeocin and neomycin, 72 surviving clones were screened that yielded three correctly targeted clones. After floxed Foxc2 mice were generated by tetraploid complementation, we removed the two selection marker genes by a simultaneous-single microinjection of expression vectors for Dre and Flp recombinases into in vitro-fertilized eggs. To delete Foxc2 in mouse kidneys, floxed Foxc2 mice were mated with Pax2-Cre mice. Newborn Pax2-Cre; Foxc2ˡᵒˣᴾ/ˡᵒˣᴾ mice showed kidney hypoplasia and glomerular cysts. These results indicate the feasibility of generating floxed Foxc2 mice by double-selection system and simultaneous removal of selection markers with a single microinjection.

A direct effect of uraemic toxins in promoting progression of chronic renal disease has not been established. In this study, we investigated the toxic effects of organic anions which characteristically appeared in the patients with progressive renal disease on renal proximal tubular cells expressing human organic anion transporter (hOAT) 1. A renal proximal tubular cell line, opossum kidney (OK) cells, was transformed with hOAT1. Among the organic anions examined, hippuric acid, para‐hydroxyhippuric acid, ortho‐hydroxyhippuric acid, indoxyl sulphate and indoleacetic acid showed a high affinity for hOAT1 expressed in the OK cells. Indoxyl sulphate and indoleacetic acid concentration‐dependently inhibited proliferation of the hOAT1‐transformed cells. The h.p.l.c. analysis demonstrated that cellular uptake of these organic anions was significantly elevated in hOAT1‐transformed cells. These organic anions also concentration‐dependently stimulated cellular free radical production. The degrees of inhibition of cell proliferation and the stimulation of free radical production induced by the organic anions were significantly higher in the hOAT1‐transformed cells than vector‐transformed cells. The stimulatory effect of indoxyl sulphate on free radical production was abolished by anti‐oxidants and probenecid. Less free radical production was observed in the hOAT1‐transformed cells treated with p‐hydroxyhippuric acid, o‐hydroxyhippuric acid compared with indoxyl sulphate and indoleacetic acid. Hippuric acid had little effect on free radical production. Organic anions present in the serum of patients with progressive renal disease may cause proximal tubular injury via hOAT1‐mediated uptake. The mechanism of cellular toxicity by these uraemic toxins involves free radical production. Thus, some uraemic toxins may directly promote progression of chronic renal disease. British Journal of Pharmacology (2002) 135, 555–563; doi:10.1038/sj.bjp.0704482

We describe the clinical ethics problem of American thyroid cancer patients being offered 'observation' instead of the USA standard of care with questionable informed consent. This problem arose because some American practitioners misinterpreted 1990s Japanese studies. American proponents of these studies failed to recognize major differences in ethical oversight between Japanese and US clinical research, misrepresenting these studies as justifying clinical practices for higher risk patients that were not supported by data. The current professional environment in American thyroid cancer management is sufficiently problematic that consideration should be made, for patients who have inadvertently consented to nonevidence-based treatments, to be recontacted and provided an opportunity to revisit their care plans or seek second opinions regarding an observational approach.

Previous surveys have suggested that elderly Japanese women have the lowest scientific interest and literacy within the Japanese population and among populations across Western countries. Because recent tremendous advances in genome analysis are likely to be incorporated into standard biomedical assessments throughout the world, we conducted surveys to investigate the attitudes toward genetic/genomic research of Japanese women aged between 55 and 65 years. Current surveys indicate that obtaining adequate informed consent from elderly Japanese women is complicated. The limitation is especially relevant to participants’ literacy in genetics and genomic studies. Results of the surveys also indicate that even after the informed consent is obtained, researchers must continue to supply updated study information to the study subjects, which enables them to obtain additional information on the use of their samples and genetic/genomic information. Failure to consider these obligations may lead to a loss of the public’s trust and thus affect research progress on medical genomics.

Uremic toxins have been suggested to promote progression of chronic renal failure by damaging tubular cells. Previous in vitro studies have indicated that some uremic toxins induce oxidative stress and activate NF-[kappa]B to upregulate plasminogen activator inhibitor-1 in tubular cells. These mechanisms may promote tubulointerstitial fibrosis. The present study examined whether uremic toxins induce glomerular and tubulointerstitial damage in vivo. Two uremic toxins, hippuric acid (HA) or indoleacetic acid (IAA), were tested in two independent experiments (HA-treated rats vs. non-HA-treated controls, IAA-treated rats vs. non-IAA-treated controls). The uremic toxins were administered to subtotally nephrectomized rats. Renal functions were measured periodically and glomerular sclerosis and interstitial fibrosis were examined at the end of the experimental period (18 and 24 weeks, respectively, after subtotal nephrectomy for HA and IAA treatments). Glomerular filtration rate (inulin clearance) at the end of the study period was significantly lower in uremic toxin-treated rats than in control rats (HA-treated rats: 0.090 + or - 0.004 ml/min/100 g body weight vs. non-HA-treated controls: 0.125 + or - 0.013, IAA-treated rats: 0.068 + or - 0.006 versus non-IAA-treated controls: 0.100 + or - 0.013; both p < 0.05). Beta-N-acetyl-glucoseamidase excretion was significantly higher in uremic toxin-treated rats than in control rats (HA-treated: 0.55 + or - 0.05 U/day vs. control: 0.39 + or - 0.04 at week 18, IAA-treated: 0.35 + or - 0.02 vs. control: 0.26 + or - 0.07 at week 16; both p < 0.05). Glomerular sclerosis index was significantly higher in uremic toxin-treated rats than in control rats (HA-treated: 0.85 + or - 0.16 versus control: 0.48 + or - 0.10, IAA-treated: 1.13 + or - 0.25 vs. control: 0.57 + or - 0.10; both p < 0.05). Significant enlargement of interstitial fibrosis was observed in indoleacetic acid-treated rats. These results indicate that overload of uremic toxins accelerates the loss of kidney function, glomerular sclerosis and tubulointerstitial injury in a rat model of chronic renal failure. The present study suggests the potential benefit of early intervention to remove various uremic toxins in delaying the onset of end-stage renal failure in patients with progressive renal disease. Copyright (C) 2003 S. Karger AG, Basel

Patients with end-stage renal disease (ESRD) develop various kinds of abnormalities in bone and mineral metabolism, widely known as renal osteodystrophy (ROD). Although the pathogenesis of ESRD may be similar in many patients, the response of the bone varies widely, ranging from high to low turnover. ROD is classified into several types, depending on the status of bone turnover, by histomorphometric analysis using bone biopsy samples [1,2]. In the mild type, bone metabolism is closest to that of persons with normal renal function. In osteitis fibrosa, bone turnover is abnormally activated. This is a condition of high-turnover bone. A portion of the calcified bone loses its lamellar structure and appears as woven bone. In the cortical bone also, bone resorption by osteoclasts is active, and a general picture of bone marrow tissue infiltration and the formation of cancellous bone can be observed. In osteomalacia, the bone surface is covered with uncalcified osteoid. This condition is induced by aluminum accumulation or vitamin D deficiency. The mixed type possesses characteristics of both osteitis fibrosa and osteomalacia. The bone turnover is so markedly accelerated that calcification of the osteoid cannot keep pace. In the adynamic bone type, bone resorption and bone formation are both lowered. While bone turnover is decreased, there is little osteoid. The existence of these various types probably accounts for the diversity in degree of renal impairment, serum parathyroid hormone (PTH) level, and serum vitamin D level in patients with ROD. However, all patients share a common factor, i.e., the presence of a uremic condition.

Uremic toxins of organic anions up-regulate PAI-1 expression by induction of NF-κB and free radical in proximal tubular cells. Uremic toxins have been suggested to promote progression of chronic renal failure. We have shown that organic anion transporter-mediated uptake of uremic toxins induces oxidative stress in opossum kidney renal tubular cells overexpressing the transporter. Plasminogen activator inhibitor-1 (PAI-1) and nuclear factor-kappa B (NF-κB) are major factors known to promote tubulointerstitial fibrosis. The present study examined the signaling pathway that is activated by uremic toxins to induce PAI-1 and activate NF-κB in human renal proximal tubular cells (HK-2). Uremic toxins in the form of organic anion were examined their ability to induce oxidative stress, PAI-1 gene expression, and NF-κB activation in HK-2. PAI-1 expression was measured by enzyme-linked immunosorbent assay (ELISA) and the Northern blotting. Human PAI-1 promoter activity was estimated by luciferase reporter gene (NKκB-luc) assay. NF-κB activation was measured by the pNFκB-luc reporter gene and electrophretic gel mobility shift assay. Among organic anion species tested, indoxyl sulfate and indoleacetic acid induced free radical production in HK-2. A nonspecific transporter inhibitor (probenecid) suppressed the IS-stimulated radical production. Indoxyl sulfate and indoleacetic acid dose dependently increased the expressions of PAI-1 mRNA and protein in these cells. The luciferase reporter gene assay revealed that indoxyl sulfate and indoleacetic acid dose dependently activated NF-κB and PAI-1 promoter. Activation of NF-κB was also confirmed by an electrophoretic gel mobility shift assay. Both antioxidant and NF-κB inhibitors dose dependently inhibited the activation of PAI-1 promoter by indoxyl sulfate. Uremic toxins induce free radical production by renal tubular cells and activate NF-κB which, in turn, up-regulates PAI-1 expression. Thus, progression of chronic renal failure may be promoted by PAI-1 up-regulation induced by uremic toxins.

Background: Composition of nonselective proteinuria includes several endogenous ligands of Toll-like receptors (TLRs) not normally present in Bowman's space, thus raising the possibility that TLRs are involved in proteinuria-mediated podocyte injury. Methods: Kidneys of NEP25 mice, a model of glomerular sclerosis induced by podocyte-specific injury, were immunohistochemically evaluated for the presence of fibrin/fibrinogen, which are potent ligands for TLRs. A podocyte cell line was treated with fibrinogen or lipopolysaccharides and examined for expression of cytokines. siRNAs were used to knockdown components of TLR signaling. Results: We found deposits of fibrin/fibrinogen only in the damaged podocytes of proteinuric kidneys, indicating that podocytes are exposed to these potent TLR ligands in proteinuric state. In cultured podocytes, we confirmed mRNA expressions of TLR2, TLR4, as well as their major TLR signal transducer, MyD88. Fibrinogen and lipopolysaccharides dose-dependently upregulated mRNA expressions of MCP-1, TNF-α and TLR2 in podocytes as well as increased the MCP-1 protein in the medium. Knockdown of TLR2 and TLR4 inhibited the fibrinogen-induced MCP-1 mRNA upregulation. Knockdown of MyD88 also inhibited the upregulation. Conclusion: These results suggest that plasma macromolecules that appear in Bowman's space in proteinuric conditions have the capacity to induce podocyte cytokines through TLRs, and thereby accelerate podocyte injury. Copyright © 2009 S. Karger AG, Basel [PUBLICATION ABSTRACT]

Background: Mice carrying the null-mutated Foxc1 gene frequently develop an anomalous double collecting system. These mice provide an ideal opportunity to specify the role of ectopic budding in the development of congenital anomalies of the kidney and urinary tract. Methods: Tissue specimens were collected from Foxc1 ch/ch mutants at several embryonic stages and at birth. The upper and lower pole kidneys were qualitatively and quantitatively examined by histology, in situ hybridization and immunohistochemistry. Results: Upper pole kidneys of newborn Foxc1 ch/ch mice were significantly more hypoplastic and contained significantly fewer glomeruli than their lower pole counterparts. On embryonic day 14.5, the stage immediately before the formation of the first urine, the upper pole kidney was already smaller than the lower pole kidney. Neither histology nor immunostaining for kidney markers showed dysplastic regions in either kidney of newborn Foxc1 ch/ch mice. Of note, expression of Foxc1 was restricted to maturing podocytes and was not detectable in any intermediate structure of nephron development in the nephrogenic zone. Conclusion: Ectopic budding alone results only in kidney hypoplasia but not dysplasia. The development of dysplasticity in the maturing kidney involves gene(s) that function beyond the initial budding stage within the metanephros.