Biomedical subjects

Enyu Imai

Publications and source records attributed to Enyu Imai.

63 records · Page 4Linked to original sources

PubMed2002

Gene expression profile of renal proximal tubules regulated by proteinuria.

BACKGROUND: Proximal tubules activated by reabsorption of protein are thought to play significant roles in the progression of kidney diseases. Thus, identification of genes related to proteinuria should provide insights into the pathological process of tubulointerstitial fibrosis. METHOD: Gene expression profiles were constructed by means of direct sequencing procedures to identify genes induced in the mouse kidney proximal tubules (PT) exposed to proteinuria. RESULTS: By comparing the gene expression of control PT to that of disease model PT, the abundantly expressed genes in control PT were down-regulated presumably because of potentially toxic effects of proteinuria. From the more than 1000 up-regulated genes, an immunity related gene, thymic shared antigen-1 (TSA-1), and a novel gene, GS188, were selected for further characterization. The increased expression of TSA-1, a member of the Ly-6 family, and of GS188 in response to proteinuria was confirmed by Northern analysis, immunohistochemistry, in situ hybridization and laser microdissection along with real-time PCR analysis. Full length cloning of GS188 identified it as a family member of LR8 that was reported to express predominantly in fibroblasts. CONCLUSIONS: The gene expression profiles showed that the expression patterns in PT were changed dramatically by proteinuria. The profiles include novel genes that should be further characterized to aid the understanding of the pathophysiology of progressive kidney diseases.

Amino Acid Sequence↗

PubMed2002

Y-27632 prevents tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction.

BACKGROUND: The small GTPase Rho is involved in cell-to-substratum adhesion and cell contraction. These actions of Rho mediated by downstream Rho effectors such as Rho-associated coiled-coil forming protein kinase (ROCK) may be partly responsible for the progression of renal interstitial fibrosis. METHODS: The anti-fibrosis effects of Y-27632, a specific ROCK inhibitor, were studied both in vivo (unilateral ureteral obstruction; UUO) and in vitro. To investigate the therapeutic efficacy of Y-27632 in UUO kidneys, smooth muscle alpha actin (SMalphaA) expression, macrophage infiltration and fibrosis in the obstructed kidneys were studied. SMalphaA, transforming growth factor beta (TGF-beta), alpha1 (I) collagen, osteopontin, macrophage chemoattractant peptide-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) gene expression were examined by Northern blotting. To elucidate the mechanism linking the Rho-ROCK pathway with renal fibrosis, the effects of Y-27632 on in vitro cell proliferation and cell migration were studied. RESULTS: In vivo analysis showed that Y-27632 suppressed SMalphaA expression, macrophage infiltration and interstitial fibrosis, and that Y-27632 suppressed SMalphaA, TGF-beta and alpha1 (I) collagen mRNA expression. In vitro analysis showed that Y-27632 did not suppress proliferation of renal fibroblasts but suppressed migration of macrophages. CONCLUSIONS: The Rho-ROCK system may play an important role in the development of tissue fibrosis, and the Rho-ROCK signaling pathway may be a new therapeutic target for preventing interstitial fibrosis in progressive renal disease.

Actins↗

PubMed2002

Tornado extraction: a method to enrich and purify RNA from the nephrogenic zone of the neonatal rat kidney.

BACKGROUND: Development of the kidney is a complicated and tightly regulated process. Although several genes responsible for the renal development have been identified to date, the precise mechanisms of spatial and temporal regulation remain to be elucidated. Therefore, expanding our knowledge of molecules that are associated with nephrogenesis will be helpful to understand the whole process. METHODS: To extract RNA selectively from the nephrogenic zone of the developing kidney, we developed a simple and reliable method. RESULTS: This method, named "tornado extraction," enriched RNA of the nephrogenic zone by about 30-fold. In combination with the suppression subtractive hybridization, a considerable number of genes that were differentially expressed in the nephrogenic zone were obtained. These genes included a series of endodermal markers such as albumin and alpha-fetoprotein as well as GDNF (glia-derived neurotrophic factor), osteoblast-specific factor-2 (OSF-2)/periostin and fetuin (one of the major serum proteins in the fetus). CONCLUSION: Tornado extraction has great value in studying genes in the nephrogenic zone of the developing kidney. Since the quality of RNA obtained by this method is excellent, tornado extraction is suitable in combination with other techniques including the subtractive hybridization method and DNA microarray analysis.

Animals↗

PubMed2002

Gene electrotransfer: potential for gene therapy of renal diseases.

The ability to pursue gene therapy has been limited by the availability of an effective and safe system for gene delivery, especially to the kidney. Electroporation is an efficient method to transfer physiologically the gene to the cells without complicated preparation. Given that the systemic delivery of the functional protein can serve for the therapy of the renal diseases, skeletal muscle targeting gene therapy might be an alternative strategy for treatment of renal disease. Gene therapy to the transplant kidney may potentially improve the graft outcome by reducing acute and chronic rejections. We review on an emerging strategy of gene electrotransfer and discuss the potential application of gene therapy to renal diseases.

Electroporation↗

PubMed2002

Quantitative analyses of osteopontin mRNA expression in human proximal tubules isolated from renal biopsy tissue sections of minimal change nephrotic syndrome and IgA glomerulonephropathy patients.

Osteopontin (OPN), a secreted phosphoprotein and chemotactic to monocytes/macrophages, is upregulated in renal cortical tubules in a variety of rodent models of renal injury and is believed to possibly have a role in tubulointerstitial injury. We previously reported the establishment of a system for the quantification of messenger RNA (mRNA) expression in isolated rat glomeruli using laser-manipulated microdissection and real-time polymerase chain reaction. This system was applied to human renal biopsy specimens. We quantified OPN mRNA expression in proximal tubules of 5 patients with minimal change nephrotic syndrome (MCNS) and 11 patients with mild immunoglobulin A (IgA) glomerulonephritis. We also examined the correlation between OPN mRNA expression in proximal tubules and clinical data and pathological findings in glomeruli and tubulointerstitial regions. Patients with MCNS showed a positive correlation between OPN mRNA expression in proximal tubules and urinary protein excretion (r = 0.93; P < 0.05), whereas for patients with IgA glomerulonephritis, logarithmic values of OPN mRNA expression in proximal tubules positively correlated with low urinary protein levels (r = 0.72; P < 0.05). Pathological changes, ranging from nonexistent to minor, in glomeruli and tubulointerstitium of these patients with mild IgA glomerulonephritis did not significantly correlate with OPN mRNA expression in proximal tubules. In patients with mild IgA glomerulonephritis, OPN mRNA expression in proximal tubules increased exponentially in response to a small amount of urinary protein (<1.2 g/d).

Adolescent↗

PubMed2002

Targeting growth factors to the kidney: myth or reality?

Growth factors and cytokines play a crucial role in the progression of renal diseases. A growing body of evidence has been obtained from experimental studies, suggesting that manipulation of the activity of growth factors and cytokines is a potential form of therapy for renal diseases. To preserve the renal function structure in progressive renal diseases, this approach is achieved by inhibition of apoptosis of renal intrinsic cells and by decrease in the fibrotic signal. Inhibition of transforming growth factor beta, platelet-derived growth factor, interleukin-1 and tumor necrosis factor alpha, and supplementation of hepatocyte growth factor, vascular endothelial growth factor and bone morphogenic protein-7 may be beneficial. Recent progress in therapeutic implements including humanized antibodies, chimeric soluble receptors, aptamers, antisense oligonucleotides, and gene therapy allow us to target the causal molecules. Administration of a combination of growth factors and cytokines is a potential therapeutic approach. Targeting signal transduction molecules and their co-factors and regulators is another possibility because the signals from various growth factors use a common pathway. Thus, targeting growth factors and cytokines in renal diseases could be a promising therapeutic approach.

Animals↗

PubMed2002

A mouse model of angiotensin II slow pressor response: role of oxidative stress.

ABSTRACT. The slow pressor response to prolonged infusions of angiotensin II (AngII) entails a delayed rise in BP. This study investigated the hypothesis that the response depends on the generation of oxidative stress. The BP and renal functional response of mice to graded doses (200, 400, and 1000 ng. kg(-1). min(-1)) of subcutaneously infused AngII was studied. The SBP of conscious mice increased by day 3 at AngII1000 but showed a delayed rise by days 9 to 13 (slow pressor response) at the lower rates of AngII infusion. By day 13, there was a graded increase in SBP with the rate of AngII infusion (Vehicle, -2.6 +/- 2.6%; AngII200, +14.1 +/- 5.0%; AngII400, +31.9 +/- 1.9%; AngII1000, +43.2 +/- 5.5%). The MAP measured under anesthesia rose significantly (P < 0.001) with AngII400 at 14 d (Vehicle, 85 +/- 2 mmHg; AngII400, 100 +/- 3 mmHg). When studied at day 6, the MAP of AngII400 rats was not elevated (88 +/- 2 mmHg; NS versus vehicle), yet the GFR was higher (1.05 +/- 0.05 versus 1.25 +/- 0.05 ml. min(-1). g(-1); P < 0.05) accompanied by an increase in the filtration fraction (FF) (28.8 +/- 1.2 versus 37.2 +/- 0.8%; P < 0.001). From day 6 through day 14, the MAP had increased (P < 0.01) in AngII400, accompanied by a significant reduction in GFR to 1.05 +/- 0.04 ml. min(-1). g(-1) (P < 0.01) and elevation of renal vascular resistance (RVR) (day 6 versus day 14, 15.3 +/- 0.6 versus 19.2 +/- 1.2 mmHg. ml(-1). min(-1). g(-1); P < 0.05). Renal excretion of 8-iso PGF(2alpha) was increased in AngII400 group at day 12 (2.52 +/- 0.35 versus 5.85 +/- 0.78 pg. day(-1); P < 0.01). The permeant superoxide dismutase mimetic tempol reduced the effects of AngII400 on the SBP (-1.7 +/- 5.8%; P < 0.01), the MAP (87 +/- 4 mmHg; P < 0.01), and the RVR (15.2 +/- 0.5 mmHg. ml(-1). min(-1). g(-1); P < 0.05) at day 14 and the renal 8-iso PGF(2alpha) excretion (3.53 +/- 0.71 pg. d(-1); P < 0.05) at day 12. It is concluded that the AngII infused mouse is a valid model for the slow pressor response. There is an early rise in GFR and FF, consistent with increased postglomerular vascular resistance and a late rise in RVR with a fall in GFR, consistent with increased preglomerular vascular resistance that is accompanied by a rise in BP. There is evidence of increased oxidative stress that is implicated in the increase in the BP and RVR in this model.

Angiotensin II↗

PubMed2001

Bone marrow is a reservoir of repopulating mesangial cells during glomerular remodeling.

The renal glomerulus, whose cellular components are developmentally derived from the mesenchyme, plays a pivotal role in filtratating plasma. Irretrievable changes of glomerular components are responsible for the initiation and progression of impaired renal function. Recently, it has been shown that functional stem cells exist in the bone marrow of adult bodies and that they can reconstitute damaged tissues of the mesenchymal origin. To examine whether the bone marrow provides stem cells to damaged glomeruli, transgenic rats carrying enhanced green fluorescence protein (EGFP rat) were established in a systemic and constitutive manner. After transplanting the bone marrow of EGFP rats into wild-type rats, the progeny of the transplanted marrow cells were tracked with a tag of EGFP. Recruitment of bone marrow-derived cells into glomeruli was dramatically facilitated in response to mesangiolysis evoked in anti-Thy1 antibody-mediated glomerulonephritis. In the restored glomeruli, 11% to 12% of glomerular cells were derived from the transplanted bone marrow. The number of bone marrow-derived CD45(+) cells transiently increased during the disease process, and CD45-negative cells constantly accounted for more than half of the bone marrow-derived population in glomeruli. Bone marrow-derived Thy1(+) cells kept increasing in number until the remodeling ceased and finally made up 7% to 8% of glomerular cells. Laser scanning microscopy displayed that the bone marrow-derived Thy1(+) cells provide structural support for glomerular capillaries, which indicates that they are mesangial cells. Although CD45(-)Thy1(-) bone marrow-derived cells exist during the remodeling of glomeruli, none of them expressed endothelial markers such as Factor VIII and RECA1 as long as they were tested. The results indicate that the bone marrow can give rise to mesangial cells in vivo.

Animals↗

PubMed2001

Electroporation-mediated gene transfer that targets glomeruli.

Electroporation has been applied to introducing DNA into several organs; however, gene expression was localized around the injected area. Examined was the efficiency of intrarenal injection of DNA followed by in vivo electroporation, using FITC-labeled oligodeoxynucleotides (FITC-ODN) and plasmid DNA expressing beta-galactosidase or luciferase. FITC-ODN or expression vectors were injected into the left renal artery; thereafter, the left kidney was electroporated between a pair of tweezer-type electrodes. FITC-ODN were transferred into all glomeruli, and transfected cells were identified as mesangial cells. Four d after transfection of the pCAGGS-LacZ gene, beta-galactosidase expression was observed in 75% of glomeruli. To compare the transfection efficacy by electroporation with that by the hemagglutinating virus of Japan (HVJ) liposome method, a luciferase reporter gene, pActLuc, was transferred into glomeruli by either electroporation or the HVJ liposome method. On day 4, electroporation resulted in higher glomerular luciferase activity than did the HVJ liposome method. We also observed that co-transfection of pcEBNA, an expression vector for Epstein-Barr virus nuclear antigen, and poriP-cLuc, oriP-harboring vector, resulted in an eightfold higher luciferase gene expression than simple poriP-cLUC: No histologic damages were seen in glomeruli or tubular epithelial cells. In conclusion, gene transfer into renal artery followed by electroporation was an effective and simple strategy for gene transfer that targets glomerular mesangial cells.

Animals↗