Application of microarray assay to nephrology.
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Animals↗
Biomedical subjects
E Imai
Publications and source records attributed to E Imai.
Transforming growth factor-beta 1 antisense oligodeoxynucleotides block interstitial fibrosis in unilateral ureteral obstruction.
BACKGROUND: Interstitial expression of transforming growth factor-beta1 (TGF-beta1) is important in tubulointerstitial fibrosis, a common process in most progressive renal diseases. However, no effective therapy for progressive interstitial fibrosis is known. Recently, we developed an artificial viral envelope (AVE)-type hemagglutinating virus of Japan (HVJ) liposome-mediated retrograde ureteral gene transfer method, which allowed us to introduce the genetic material selectively into renal interstitial fibroblasts. METHOD: We introduced antisense or scrambled oligodeoxynucleotides (ODNs) for TGF-beta 1 into interstitial fibroblasts in rats with unilateral ureteral obstruction, a model of interstitial fibrosis, to block interstitial fibrosis by retrograde ureteral injection of AVE-type HVJ liposomes. RESULTS: TGF-beta 1 and type I collagen mRNA increased markedly in the interstitium of untreated obstructed kidneys, and those were not affected by scrambled ODN transfection. Northern analysis and in situ hybridization revealed that the levels of TGF-beta 1 and type I collagen mRNA were dramatically decreased in antisense ODN-transfected obstructed kidneys. Consequently, the interstitial fibrotic area of the obstructed kidneys treated with antisense ODN was significantly less than that of the obstructed kidneys untreated or treated with scrambled ODN. CONCLUSION: The introduction of TGF-beta 1 antisense ODN into interstitial fibroblasts may be a potential therapeutic maneuver for interstitial fibrosis.
Actins↗
Calcium channel blockers inhibit proliferation and matrix production in rat mesangial cells: possible mechanism of suppression of AP-1 and CREB activities.
BACKGROUND: Calcium channel blockers (CCBs) are reported to attenuate the loss of renal function in various glomerulonephritides. METHODS: To determine the mechanism of action of these drugs, we investigated the effects of CCBs on cell proliferation and extracellular matrix (ECM) production in cultured rat mesangial cells. RESULTS: While stimulation with 5% fetal calf serum (FCS) increased [(3)H]thymidine and [(3)H]proline incorporation into quiescent mesangial cells, incubation with nifedipine and cilnidipine inhibited the increase in a dose-dependent manner. Northern blot analysis demonstrated that 5% FCS increased the expression of transforming growth factor beta (TGF-beta) and fibronectin (FN) mRNA and that CCBs significantly reduced this induction, indicating that CCBs may reduce ECM production through inhibiting TGF-beta and FN. Since activator protein 1 (AP-1) regulates cell proliferation and TGF-beta expression, we evaluated the AP-1 activity by gel mobility shift analysis. Nuclear extracts of FCS-treated cells showed a strong binding to AP-1-specific oligonucleotides which was suppressed by CCBs, suggesting that these agents may inhibit cell proliferation by suppressing AP-1. CCBs also inhibited the binding activity of cyclic adenosine monophosphate responsive element binding protein which regulates FN gene expression. However, neither CCBs nor FCS affected the NFkappaB activity. CONCLUSION: These results suggest that CCBs may, in part, inhibit the progression of glomerulonephritis through non-hemodynamic actions that include the suppression of mesangial cell proliferation and the production of ECM.
Animals↗
Gene therapy for kidney disease.
Gene therapy has distinct potential to treat disease at the most fundamental level. However, the ability to pursue gene therapy for renal disease has been limited by the availability of an adequate system for gene delivery to the kidney and for regulation of transgene expression. Presently, there are several limitations to overcome before clinical use of viral vector systems for targeting kidney can be considered. Non-viral vectors such as haemagglutinating virus of Japan (HVJ)-liposome mediated gene transfer and cationic liposome are promising but need to be improved. Given that the systemic delivery of the functional protein can serve as therapy for the renal diseases, skeletal muscle targeting gene therapy might be an alternative strategy for the treatment of renal disease. Gene therapy to the transplant kidney may potentially improve the graft outcome by reducing acute and chronic rejection. We review emerging strategies of gene transfer with reference to the kidney and discuss the potential application of gene therapy to renal diseases.
Animals↗
Transfection of rat kidney with human 15-lipoxygenase suppresses inflammation and preserves function in experimental glomerulonephritis.
The human 15-lipoxygenase (15-LO) gene was transfected into rat kidneys in vivo via intra-renal arterial injection. Three days later, acute (passive) or accelerated forms of antiglomerular basement membrane antibody-mediated glomerulonephritis were induced in transfected and nontransfected or sham-transfected controls. Studies of glomerular functions (filtration and protein excretion) and ex vivo glomerular leukotriene B(4) biosynthesis at 3 hr, and up to 4 days, after induction of nephritis revealed preservation or normalization of these parameters in transfected kidneys that expressed human 15-LO mRNA and mature protein, but not in contralateral control kidneys or sham-transfected animals. The results provide in vivo-derived data supporting a direct anti-inflammatory role for 15-LO during immune-mediated tissue injury.
Animals↗
[Tubulo-interstitial nephropathies caused by electrolyte disturbances].
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Humans↗
Elongation of oligopeptides in a simulated submarine hydrothermal system.
Oligomerization of a peptide was attempted in a flow reactor that simulated a submarine hydrothermal system. When fluid containing glycine repeatedly circulated through the hot and cold regions in the reactor, oligopeptides were made from glycine. When divalent ions (such as copper ions) were added under acidic conditions, oligoglycine was elongated up to hexaglycine. This observation suggests that prebiotic monomers could have oligomerized in the vicinity of submarine hydrothermal vents on primitive Earth.
Chromatography, High Pressure Liquid↗
Autocatalytic synthesis of oligoglycine in a simulated submarine hydrothermal system.
A flow reactor simulating a submarine hydrothermal system was constructed for examining the likelihood of oligopeptide synthesis from amino acids alone without recourse to any of condensing agents, templates or metallic ions. We demonstrated that when the initial reactant is glycine, the flow reactor can synthesize both di- and tri-glycine. The initial buildup of the yields of both the oligopeptides was found to be exponential with the elapse of time. The oligopeptide synthesis from glycine in the flow reactor could be autocatalytic.
Catalysis↗
Gene therapy by transforming growth factor-beta receptor-IgG Fc chimera suppressed extracellular matrix accumulation in experimental glomerulonephritis.
BACKGROUND: The evidence that transforming growth factor-beta (TGF-beta) is a key mediator in the pathogenesis of fibrotic diseases is now supported by several lines of investigation. This evidence provides a certain base for targeting TGF-beta as an antifibrotic agent. METHODS: We generated a chimeric cDNA, termed TGF beta RII/Fc, encoding an extracellular domain of the TGF-beta type II receptor fused to the IgG-Fc domain, and tested whether TGF beta RII/Fc could be a novel strategy for treating glomerular diseases. RESULTS: In cultured BNul-7 cells, recombinant TGF beta RII/Fc reversed the antiproliferative response induced by TGF-beta 1. In addition, TGF beta RII/Fc diminished the TGF-beta 1-induced production of EIIIA-positive fibronectin in cultured normal rat kidney cells. We then introduced the chimeric cDNA into the muscle of the nephritic rats by the hemagglutinating virus of Japan liposome-mediated gene transfer method in order to block the TGF-beta activity in nephritic glomeruli through systemic delivery of chimeric molecules. Treatment with TGF beta RII/Fc gene transfection could suppress the glomerular TGF-beta mRNA in nephritic rats with a comparable effect in the reduction of extracellular matrix accumulation. CONCLUSION: TGF beta RII/Fc successfully inhibited the action of TGF-beta in vitro and in vivo, and gene therapy by chimeric TGF beta RII/Fc might be feasible for the therapy of glomerulosclerosis.
Animals↗
High water intake ameliorates tubulointerstitial injury in rats with subtotal nephrectomy: possible role of TGF-beta.
BACKGROUND: It has been shown that tubulointerstitial injury correlates well with a decline of renal function. In this study, we investigated the effect of high water intake (HWI) on functional and structural parameters in rats with subtotal nephrectomy. METHODS: Two weeks after the ablative procedure, rats were divided into two groups. One group received the treatment with HWI (3% sucrose added to drinking water) for eight weeks. Functional parameters were compared with sham-operated control (CONT) or nephrectomized rats without treatment (NX). Remnant kidneys were then assessed histologically for evidence of interstitial fibrosis and glomerulosclerosis. RESULTS: Creatinine clearance was significantly improved in HWI rats compared with NX rats. Simultaneously, urinary protein was also significantly reduced in HWI rats. HWI predominantly ameliorated interstitial lesions and, to a lesser extent, glomerular lesions. Northern blot analysis demonstrated that transforming growth factor-beta (TGF-beta) mRNA expression was significantly suppressed in HWI rats. In situ hybridization revealed that HWI suppressed TGF-beta mRNA expression mainly in the outer medulla. Fibronectin mRNA was also reduced by the HWI treatment. The changes in TGF-beta and fibronectin mRNA were in parallel with Na+/myo-inositol cotransporter (SMIT) mRNA, which is regulated by extracellular osmolarity. Immunohistochemistry demonstrated that protein expression of TGF-beta and fibronectin coincided with the mRNA expression. CONCLUSION: These results suggest that HWI reduces TGF-beta mRNA expression in medullary interstitium and ameliorates tubulointerstitial injury in rats with reduced renal mass.
Animals↗
Role of intron 1 in smooth muscle alpha-actin transcriptional regulation in activated mesangial cells in vivo.
BACKGROUND: The activation of glomerular mesangial cells is one of the early, important features of progressive glomerular disease. Smooth muscle alpha-actin (SMalphaA) is an excellent marker of activated mesangial cells. However, the mechanisms of SMalphaA regulation are only available from in vitro investigation. METHODS: We examined in vivo promoter analysis of the SMalphaA gene-utilizing transgenic mice harboring different promoter regions of the SMalphaA gene fused to chloramphenicol acetyl transferase (CAT). CAT activities were tested in primary cultured mesangial cells and in glomerular legions of Habu venom glomerulonephritis. RESULTS: The DNA sequence -891 to +3828, which contains exon 1, intron 1, and the first 14 bp of exon 2 in addition to the 5'-flanking sequence of the SMalphaA gene, induced high levels of transcription in activated mesangial cells in in vivo habu venom glomerulonephritis and in cultured mesangial cells derived from transgenic mice. The DNA region -891 to -124 was a positive element in mesangial cells derived from transgenic mice. Deletions (3316 or 137 bp) in intron 1 reduced transcription to undetectable levels. The 137 bp sequence is highly conserved among several species, containing one CArG box element, which is one of the key motifs for transcriptional activation of contractile-related proteins. In vitro transfection analysis failed to demonstrate these positive effects of intron 1 and region -891 to -124. Conclusions. In vivo promoter analysis of the SMalphaA gene provided new information about the transcriptional regulation of SMalphaA in activated mesangial cells. The DNA region -891 to -124 has a positive effect on SMalphaA transcription in cultured mesangial cells. The intron 1 region (+1088 to +1224) plays a pivotal role in SMalphaA transcription in activated mesangial cells in vivo. Further analysis of this conserved region in intron 1, including the CArG motif, will be of great value in understanding the molecular mechanisms of mesangial activation.
Actins↗
Increased oxidative stress in mouse kidneys with unilateral ureteral obstruction.
BACKGROUND: Unilateral ureteral obstruction (UUO) is a well-established experimental model of renal injury leading to interstitial fibrosis. The molecular and cellular mechanism(s) of interstitial fibrosis in UUO kidney is beginning to be elucidated. Oxidative stress has been implicated in the pathogenesis of various forms of renal injury; however, little is known about its involvement in the setting of ureteral obstruction. METHODS: To investigate the possible involvement of oxidative stress in the obstructive nephropathy, we studied the occurrence and distribution of Nepsilon-carboxymethyl-lysine (CML) in the kidneys after ureteral obstruction. CML is an integrative biomarker of the cumulative protein damage induced by glycoxidation. Heme oxygenase-1 (HO-1) mRNA and protein expression, which is a sensitive and reliable indicator of oxidative stress, were also examined. RESULTS: CML immunoreactivity was found in the interstitium of UUO kidneys 10 days after the onset ureteral obstruction. HO-1 mRNA was up-regulated as early as 12 hours after ureteral obstruction. HO-1 immunoreactivity was observed in the periglomerular and peritubular interstitium two days after ureteral obstruction. CONCLUSIONS: These results strongly suggested the presence of increased oxidative stress in the interstitium of UUO kidneys. The oxidative stress and the formation of various kind of biological active oxidative products in the interstitium are supposed to play significant roles in UUO kidney.
Animals↗
Low density lipoprotein apheresis therapy for steroid-resistant nephrotic syndrome. Kansai-FGS-Apheresis Treatment (K-FLAT) Study Group.
BACKGROUND: The pathogenic role of hyperlipidemia in long-standing nephrotic syndrome (NS) is known to be responsible for both the progression of glomerulosclerosis and tubulointerstitial injury, especially in focal segmental glomerulosclerosis (FGS). METHODS: Aggressive lipid lowering treatment by low density lipoprotein (LDL) apheresis (LDL-A) using a dextran sulfate cellulose column to treat patients with steroid-resistant or frequently recurrent severe NS was performed first without fixing the protocol in eight patients with FGS and one with minimal change nephrotic syndrome (MCNS). The period of NS before LDL-A, number and average intervals of LDL-A until the end of the therapy, and the prognosis were investigated. Next, a multicenter study with a fixed protocol of LDL-A treatment was designed in combination with steroid therapy for treatment twice a week for three weeks and weekly for six weeks, and was performed in 17 patients with FGS. The effects on the state of NS in addition to the change of urinary eicosanoid metabolites and remission rates were evaluated. RESULTS: In the preliminary study, along with a rapid improvement of hyperlipidemia, a high incidence of remission was achieved by LDL-A performed at relatively short intervals. In the multicenter study with a fixed protocol, there was a significant decrease of urinary protein (P < 0.001) and increase of serum albumin (P < 0.02) as well as a decrease of thromboxane B2 (TXB2) excretion (P < 0.05) after the treatment. Urinary excretion of TXB2 was significantly reduced after LDL-A (P < 0.05). The rate of entering into complete or incomplete remission was 71% with a relatively short duration of nephrotic-range proteinuria using the LDL-A therapy in comparison with steroid therapy alone. CONCLUSION: The rapid improvement of hypercholesterolemia with LDL-A treatment may provide a new approach for a high rate of improvement in the degree of NS in steroid-resistant NS of FGS and MCNS.
Adolescent↗
Probucol suppresses ICAM-1 expression in rat mesangial cells: possible role of IL-1.
Interleukin-1 (IL-1) participates in the progression of glomerulonephritis by up-regulating intercellular adhesion molecule-1 (ICAM-1) expression in experimental glomerulonephritis. Probucol, an anti-hyperlipidemic agent, ameliorates some types of glomerulonephritis regardless of serum cholesterol levels, and is also reported to inhibit IL-1 release from macrophages in atherosclerotic lesions. However, little is known about the mechanism of this favorable action on glomerular injury. We examined whether or not probucol inhibits ICAM-1 expression by suppression of IL-1 action in cultured rat mesangial cells. In this brief report, we review the actions of probucol on IL-1 secretion and discuss the possible mechanism by which probucol may suppress the glomerular injury.
Animals↗
Response to hypertonicity in mesothelial cells: role of Na+/myo-inositol co-transporter.
BACKGROUND: During peritoneal dialysis, the peritoneal mesothelium is exposed continually to hypertonic dialysates. The purpose of this study is to see if rat mesothelial cells have an osmoregulatory mechanism to adapt to hypertonic environment. METHODS: The intracellular content of organic osmolytes was measured by HPLC methods. Myo-inositol transport activity was measured by Na+-dependent uptake of [3H]myo-inositol. mRNA abundance for the Na+/myo-inositol co-transporter (SMIT) was examined by Northern and slot-blot analyses. RESULTS: In isotonic mesothelial cells, only myo-inositol could be detected. After switching to hypertonic medium made by addition of NaCl, myo-inositol content gradually increased and peaked at 48 h after the switch. The myo-inositol content in hypertonic cells increased > 7-fold over the value in isotonic cells. The contents of betaine and glycerophosphorylcholine (GPC) also increased but were less than that of myo-inositol. Sorbitol was not accumulated in this condition. When glucose was used to increase medium osmolality, all of the four osmolytes were increased by hypertonicity (myo-inositol > sorbitol > GPC > betaine). Thus, myo-inositol is the most abundant osmolyte in the mesothelial cells. Na+-dependent myo-inositol uptake in hypertonic cells was approximately 7-fold the uptake in isotonic cells, reaching a maximum 16 h after switching to a hypertonic medium. The uptake rate increased as medium osmolality increased from 300 to 500 mosm/kg. SMIT mRNA rapidly increased after increasing medium osmolality, reaching a maximum 8 h after the switch. The relative increase in the mRNA abundance was approximately 11 times isotonic levels. CONCLUSIONS: Mesothelial cells respond to extracellular hypertonicity by increasing SMIT mRNA abundance, myo-inositol transport activity and accumulating myo-inositol into the cells.
Animals↗
Enhanced interstitial expression of caldesmon in IgA nephropathy and its suppression by glucocorticoid-heparin therapy.
BACKGROUND: With progressive renal disease, structural derangement increasingly encompasses the tubulointerstitial compartment. Tubulointerstitial injury is a critical determinant of renal functional reserve and prognosis in renal disease. Interstitial cells acquiring characteristic of myofibroblasts are an important contributor to interstitial fibrosis. Caldesmon, a calmodulin or actin binding protein, is a molecular marker of differentiation in smooth muscle cells and has recently been shown by us to be a good marker of mesangial cell activation in IgA nephropathy patients. METHODS. We studied whether the expression of caldesmon in interstitium of the kidney was enhanced in the process of glomerular disease and whether it would be a marker of interstitial activation in specific disease states. We performed immunohistochemical staining with anti-caldesmon antibodies in 38 biopsy specimens from IgA nephropathy patients and analysed them quantitatively with a computer-aided manipulator. Interstitial caldesmon expression were compared with histological changes and clinical parameters. RESULTS: Caldesmon expression was enhanced where interstitial cell infiltration and fibrosis were found. Immunoelectron microscopy revealed that caldesmon staining in the renal interstitium was cytoplasmic, and in the processes of myofibroblast-like cells. Caldesmon expression was more prominent in the intense CD68 infiltrated group than in the low positive cells infiltrated group. Patients showing high intensity of interstitial caldesmon expression had significantly higher urinary protein excretion than those showing low intensity of caldesmon expression. Next, 15 patients were treated with glucocorticoid and heparin for 4-8 weeks and re-biopsies were performed. Caldesmon expression was reduced in concomitant with decreased interstitial cell infiltration. Follow-up of these patients (average 24 months) revealed a significant suppression of urinary protein excretion and significant improvement of creatinine clearance. CONCLUSION: These results suggest that the interstitial caldesmon expression is associated with the progression of IgA nephropathy, and glucocorticoid--heparin therapy may reverse the phenotypic change of interstitial cells during the disease process of glomerulonephritis.
Actins↗
Cytokines and glomerulosclerosis.
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Animals↗
New paradigm of gene therapy: skeletal-muscle-targeting gene therapy for kidney disease.
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Animals↗