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Michio Kuwahara

Publications and source records attributed to Michio Kuwahara.

At least 19 recordsLinked to original sources
PubMed2005

Water channel activities of Mimosa pudica plasma membrane intrinsic proteins are regulated by direct interaction and phosphorylation.

cDNAs encoding aquaporins PIP1;1, PIP2;1, and TIP1;1 were isolated from Mimosa pudica (Mp) cDNA library. MpPIP1;1 exhibited no water channel activity; however, it facilitated the water channel activity of MpPIP2;1 in a phosphorylation-dependent manner. Mutagenesis analysis revealed that Ser-131 of MpPIP1;1 was phosphorylated by PKA and that cooperative regulation of the water channel activity of MpPIP2;1 was regulated by phosphorylation of Ser-131 of MpPIP1;1. Immunoprecipitation analysis revealed that MpPIP1;1 binds directly to MpPIP2;1 in a phosphorylation-independent manner, suggesting that phosphorylation of Ser-131 of MpPIP1;1 is involved in regulation of the structure of the channel complex with MpMIP2;1 and thereby affects water channel activity.

Animals↗
PubMed2005

Differential regulation of AQP2 trafficking in endosomes by microtubules and actin filaments.

Vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct cells is critical to regulate the urine concentration. To better understand the mechanism of subcellular trafficking of AQP2, we examined MDCK cells expressing AQP2 as a model. We first performed double-immunolabeling of AQP2 with endosomal marker proteins, and showed that AQP2 is stored at a Rab11-positive subapical compartment. After the translocation to the plasma membrane, AQP2 was endocytosed to EEA1-positive early endosomes, and then transferred back to the original Rab11-positive compartment. When Rab11 was depleted by RNA interference, retention of AQP2 at the subapical storage compartment was impaired. We next examined the role of cytoskeleton in the AQP2 trafficking and localization. By the treatment with microtubule-disrupting agent such as nocodazole or colcemid, the distribution of AQP2 storage compartment was altered. The disruption of actin filaments with cytochalasin D or latrunculin B induced the accumulation of AQP2 in EEA1-positive early endosomes. Altogether, our data suggest that Rab11 and microtubules maintain the proper distribution of the subapical AQP2 storage compartment, and actin filaments regulate the trafficking of AQP2 from early endosomes to the storage compartment.

Actin Cytoskeleton↗
PubMed2005

Role of the E2F1-p19-p53 pathway in ischemic acute renal failure.

BACKGROUND: Cell cycle progression and arrest of renal tubular cells after acute injury is a reactive process of renal regeneration. The p16(INK4a)/p19(ARF) (alternative reading frame) locus encodes two proteins involved in cell cycle regulation. We investigated the transcriptional regulation and tissue distribution of p19(ARF) in ischemic acute renal failure (ARF). METHODS: We examined the time course and immunohistochemistry of p19(ARF) in rat kidneys following the induction of ischemic ARF. We also examined the effect of p19(ARF) overexpression on p53 levels and cell cycle progression in MDCK cells. RESULTS: The protein expression of p19(ARF) strongly increased 72 h after the ischemia. Immunohistochemical studies showed that the renal tubular cells in the outer medulla expressed p19(ARF) protein 72 h after ischemic injury. The time course of E2F1 induction was observed at 6-24 h, and it was found to precede p19(ARF) expression. In MDCK cells, the overexpression of E2F1 increased promoter activity and the protein level of p19(ARF) and induced apoptosis. Transfection of the p19(ARF) expression vector caused an increase in p53 protein, cell cycle arrest and apoptosis. CONCLUSIONS: These data support the hypothesis that the E2F1-p19(ARF)-p53 pathway forms a negative feedback loop to regulate the cell cycle of renal tubular cells in the ischemic ARF.

Acute Kidney Injury↗
PubMed2005

Aldosterone stimulates proliferation of mesangial cells by activating mitogen-activated protein kinase 1/2, cyclin D1, and cyclin A.

Recently, attention has been focused on the role of aldosterone in the pathophysiology of hypertension and cardiovascular disease. Several clinical and experimental data support the hypothesis that aldosterone contributes to the progression of renal injury. However, the molecular mechanisms of the effects of aldosterone in signal transduction and the cell-cycle progression of mesangial cells are not well known. For determining the signaling pathway of aldosterone in cultured mesangial cells, the effects of aldosterone on the mitogen-activated protein kinase 1/2 (MAPK1/2) pathway and the promoter activities of cyclin D1, cyclin A, and cyclin E were investigated. First, it was shown that the mineralocorticoid receptor (MR) was expressed in rat mesangial cells and glomeruli and that aldosterone stimulated the proliferation of mesangial cells via the MR and MAPK1/2 pathway. Next, it was demonstrated that aldosterone stimulated Ki-RasA, c-Raf kinase, MEK1/2, and MAPK1/2 in rat mesangial cells. Aldosterone induced cyclin D1 and cyclin A promoter activities and protein expressions, as well as the increments of CDK2 and CDK4 kinase activities. The presence of CYP11B2 and 11beta-HSD2 mRNA in rat mesangial cells also was shown. In conclusion, aldosterone seems to exert mainly MR-induced effects that stimulate c-Raf, MEK1/2, MAPK1/2, the activities of CDK2 and CDK4, and the cell-cycle progression in mesangial cells. MR antagonists may serve as a potential therapeutic approach to mesangial proliferative disease.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗
PubMed2005

Identification of a novel aquaporin, AQP12, expressed in pancreatic acinar cells.

Members of the aquaporin (AQP) water channel family are widely distributed in various tissues and contribute to the water permeability of epithelial and endothelial cells. Currently 11 members of the AQP family (AQP0-10) have been reported in mammals. Here we report the identification of AQP12, which we found by performing a BLAST program search. Northern blot analysis revealed that AQP12 was specifically expressed in the pancreas. Further analysis by in situ hybridization and RT-PCR studies showed that AQP12 was selectively localized in the acinar cells of the pancreas. To analyze the cellular localization and function of AQP12, we expressed AQP12 in Xenopus oocytes and cultured mammalian cells. Immunocytochemistry revealed that AQP12 was not targeted to the plasma membrane. The selective localization of AQP12 in pancreatic acinar cells and possibly in the intracellular organelles suggests a role of AQP12 in digestive enzyme secretion such as maturation and exocytosis of secretory granules.

Amino Acid Sequence↗
PubMed2005

The C-terminal tail of aquaporin-2 determines apical trafficking.

BACKGROUND: Aquaporin-2 (AQP-2) proteins are mainly expressed at the apical region of the collecting duct cells. We previously reported three different mutations in the C-terminus of AQP-2 that all-cause autosomal-dominant nephrogenic diabetes insipidus. When one of these mutant AQP-2s was expressed in Madin-Darby canine kidney (MDCK) cells, it was mistargeted to the basolateral membrane, suggesting a critical role of the C-terminal tail in the apical trafficking of AQP-2. METHODS: Portions of the AQP-2 C-terminal tail (residues 226-271) were mutated by the polymerase chain reaction (PCR) technique and inserted into the pcDNA3.1 vector. Constructs were transfected into MDCK cells to examine the localization of mutated AQP-2 proteins by immunofluorescence microscopy. Cell surface expression was detected by biotinylation assay. RESULTS: The wild-type AQP-2 was localized at the apical membrane, whereas mutants lacking residues 262-271 (the last 10 amino acids) were predominantly distributed in the endoplasmic reticulum. Deletion mutants of the initial (226-240del) and middle (241-252del) portions of the C-terminal tail were identified at the apical membrane, suggesting that residues 226-252 have no involvement in apical targeting. An AQP-4-AQP-2 chimera in which a portion of the AQP-4 C-terminal tail was replaced by the corresponding site in AQP-2 (residues 256-271) was found at the apical membrane. The sequence of the last 4 amino acids of AQP-2 (G-T-K-A) corresponds to a PDZ-interacting motif. Our investigations identified a mutant of this portion mostly localized to the subapical region. Further, apical expression was found to be significantly decreased in mutants lacking a consensus sequence for cyclic adenosine monophosphate (cAMP)-dependent phosphorylation (residues 253-256). CONCLUSION: The sequence at 256-271 is sufficient for apical trafficking in AQP-2. The putative PDZ-interacting motif (G-T-K-A, residues 268-271) plays a key role in apical membrane expression. In addition, cAMP-dependent phosphorylation was found to be critical for apical targeting.

Amino Acid Sequence↗
PubMed2005

[Idiopathic edema].

Idiopathic edema is an orthostatic edema accompanied by excessive weight gain from morning to evening. It occurs almost exclusively in post pubertal women and is independent of menstral cycle. Although the cause is not clear, a number of hormonal abnormalities are postulated to be involved. These include rennin-angiotensin-aldosterone axis, vasopressin, atrial natriuretic factor, dopamine, hypothalamic disorders, and thyroid disorders. In addition, exaggerated postual abnormalities, psychological or emotional disturbance, eating disorders, and abuse of diuretic or laxative are also associated with the onset of edema.

Edema↗
PubMed2004

Aquaporin-2 trafficking is regulated by PDZ-domain containing protein SPA-1.

Targeted positioning of water channel aquaporin-2 (AQP2) strictly regulates body water homeostasis. Trafficking of AQP2 to the apical membrane is critical to the reabsorption of water in renal collecting ducts. Controlled apical positioning of AQP2 suggests the existence of proteins that interact with AQP2. A biochemical search for AQP2-interacting proteins led to the identification of PDZ-domain containing protein, signal-induced proliferation-associated gene-1 (SPA-1) which is a GTPase-activating protein (GAP) for Rap1. The distribution of SPA-1 coincided with that of AQP2 in renal collecting ducts. The site of colocalization was concomitantly relocated by hydration status. AQP2 trafficking to the apical membrane was inhibited by the SPA-1 mutant lacking Rap1GAP activity and by the constitutively active mutant of Rap1. AQP2 trafficking was impaired in SPA-1-deficient mice. Our results show that SPA-1 directly binds to AQP2 and regulates at least in part AQP2 trafficking.

Animals↗
PubMed2004

Aquaporin-2 is retrieved to the apical storage compartment via early endosomes and phosphatidylinositol 3-kinase-dependent pathway.

Aquaporin-2 (AQP2) is one of the water-channel proteins expressed in principal cells of kidney collecting ducts, where it is stored in the intracellular compartment. Previous studies have demonstrated that AQP2 vesicles constitute a distinct intracellular compartment partially overlapping with early endosomes. In this report, we performed in vitro experiments using the renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, stably expressing AQP2 (MDCK-hAQP2). In nonpolarized cells, AQP2 vesicles were scattered in the cytoplasm and did not colocalize with Golgi 58K or TGN38. Small portions of AQP2 vesicles were positive for the lysosome marker cathepsin D. An early endosome antigen (EEA1) localized around AQP2 vesicles in close proximity, suggesting involvement of the endosomal system in the trafficking of AQP2. AQP2 vesicles are distinct from other recycling molecules, such as glucose transporter 4 (GLUT4) and endocytosed transferrin. In polarized MDCK-hAQP2 cells, AQP2 vesicles were localized in the subapical recycling compartment and distinct from the Golgi apparatus, trans-Golgi network, lysosome, and early endosome in the nonstimulated state. When the cells were treated with forskolin, translocation of AQP2 to the apical membrane was observed. Washout of forskolin induced retrieval of AQP2 into the cytoplasm, and AQP2 was transiently colocalized with EEA1-positive endosomes. Then, AQP2 moved from EEA1-positive endosomes to the subapical AQP2-storage compartment, which is sensitive to wortmannin and LY294002. These results suggest that AQP2 resides in a recycling compartment at the apical side in polarized MDCK-hAQP2 cells, and its retrieval uses the apical endosomal system and the phosphatidylinositol 3-kinase-dependent pathway.

Amino Acid Sequence↗
PubMed2004

Expression and function of Ets-1 during experimental acute renal failure in rats.

The Ets family of transcription factors is defined by a conserved DNA-binding Ets domain that forms a winged helix-turn-helix structure motif. The Ets family is involved in a diverse array of biologic functions, including cellular growth, migration, and differentiation. The hypothesis in this study was that Ets-1 is re-expressed during regeneration after acute renal failure (ARF) and plays a key role in the transcriptional regulation of cyclin D1 and the cell cycle progression in renal tubular cells. For clarifying the significance of Ets-1 in ARF, a rat ARF model in vivo and LLC-PK1 cells as an in vitro model were used. After the left rat renal artery was clamped for 1 h, the whole kidney homogenate was examined and total RNA was extracted at 6, 12, 24, 48, and 72 h after reperfusion by Western blot analysis and real-time reverse transcription-PCR. Ets-1 mRNA and protein expression were strongly increased at 6 to 24 h after the ischemia, respectively. The expression of hypoxia-inducible factor-1alpha was increased dramatically as early as 6 h after ischemia-reperfusion and decreased at 48 and 72 h after ischemia-reperfusion. In the immunohistologic examination, Ets-1 was expressed in the proximal tubules and coexpressed with proliferating cell nuclear antigen (PCNA). Furthermore, overexpression of Ets-1 promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Ets-1 promoter activity increased between 3 and 6 h in hypoxia, and hypoxia also induced changes in the Ets-1 protein level in LLC-PK1 cells. The Ets-1 induction by hypoxia was abolished by the transfection of dominant-negative hypoxia-inducible factor-1alpha. A gel shift assay demonstrated that Ets-1 binds to the ets-1 binding site of the cyclin D1 promoter in the ischemia-reperfusion condition. Overexpression of Ets-1 did not significantly change the caspase 3 activity or the value of cell death ELISA in LLC-PK1 cells. Taken together, these data suggest that Ets-1 plays a key role in the cell-cycle progression of renal tubules in ARF. The Ets-1 pathway may regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF.

Acute Kidney Injury↗
PubMed2004

Renoprotective effect of losartan in comparison to amlodipine in patients with chronic kidney disease and hypertension--a report of the Japanese Losartan Therapy Intended for the Global Renal Protection in Hypertensive Patients (JLIGHT) study.

A 12-month, multicenter (57 clinical institutions), randomized, open-labeled trial was undertaken to compare the efficacy of the angiotensin II receptor antagonist losartan and the calcium channel blocker amlodipine in patients with proteinuric chronic kidney disease (CKD) and hypertension. A total of 117 patients (79, chronic glomerulonephritis; 14, diabetic nephropathy; 24, other CKD) were randomly allocated into two treatment groups. Losartan and amlodipine exerted the same efficacy for blood pressure (BP) control; however, losartan significantly reduced the 24-h urinary protein excretion at months 3, 6, and 12, with the reduction of 20.7%, 35.2%, 35.8%, whereas amlodipine did not change the amount of proteinuria over the 12-month study period. When patients were stratified into groups according to the level of BP control at 3 months, the reduction in urinary protein excretion by losartan was evident in the group for which a BP of <140/90 mmHg was achieved, as well as in the group for which the goal BP (<130/85 mmHg) for treatment of CKD was not achieved. When patients were stratified according to baseline urinary protein excretion, those with > or = 2 g/day showed a reduction in proteinuria by losartan of 23.3%, 39.4%, and 47.9% at months 3, 6, and 12, and those with <2 g/day showed a reduction of 18.5% and 31.2% at months 3 and 6, respectively. No fatal adverse events were experienced in either drug group. We conclude that losartan reduced proteinuria in patients with CKD and hypertension. This positive effect may contribute to the renal protective benefit of losartan, and is beyond the magnitude of BP control.

Aged↗
PubMed2003

Interim evidence of the renoprotective effect of the angiotensin II receptor antagonist losartan versus the calcium channel blocker amlodipine in patients with chronic kidney disease and hypertension: a report of the Japanese Losartan Therapy Intended for Global Renal Protection in Hypertensive Patients (JLIGHT) Study.

BACKGROUND: Insufficiency of renal function and high blood pressure influence each other and eventually result in life-threatening endstage renal disease. It has been proposed that proteinuria per se is a determinant of the progression of chronic kidney disease (CKD). The therapeutic strategy for patients with proteinuric CKD and hypertension should therefore be targeted with a view not merely toward blood pressure reduction but also toward renoprotection. METHODS: We examined the effect of the angiotensin (AT)1 receptor antagonist losartan and the calcium channel blocker amlodipine, throughout a period of 12 months, on reduction of blood pressure and renoprotection. This was done by assessing amounts of urinary protein excretion, serum creatinine (SCr), and creatinine clearance (CCr) in patients with hypertension (systolic blood pressure [SBP] > or = 140 mmHg or diastolic blood pressure [DBP] > or = 90 mmHg) and CKD (male, body weight [BW] > or = 60 kg: 1.5 < or = SCr < 3.0 mg/dl; female or male BW < 60 kg: 1.3 < or = SCr < 3.0 mg/dl), manifesting proteinuria of 0.5 g or more/day. Losartan was administered once daily at doses of 25 to 100 mg/day, and amlodipine was given once daily at 2.5 to 5 mg/day. No antihypertensive combination therapy was allowed during the first 3-month period. RESULTS: A 3-month interim analysis revealed that, despite there being no difference in blood pressure between the two groups, there was a significant reduction in 24-h urinary protein excretion in the losartan group ( n = 43), but there was no change in the amlodipine group ( n = 43). Analysis of stratified subgroups with proteinuria of 2 g or more/day and less than 2 g/day showed that losartan lowered proteinuria by approximately 24% in both subgroups, while amlodipine lowered proteinuria by 10%, but only in the subgroup of less than 2 g/day (NS). SCr and CCr did not change throughout the period of 3 months in either group. No severe or fatal adverse event was experienced in either group during the study period. CONCLUSIONS: Losartan appeared to be efficacious for renoprotection in patients with proteinuric CKD and hypertension, with the mechanism being independent of its antihypertensive action.

Adult↗
PubMed2003

Pathogenesis of nephrogenic diabetes insipidus by aquaporin-2 C-terminus mutations.

BACKGROUND: We previously reported three aquaporin-2 (AQP2) gene mutations known to cause autosomal-dominant nephrogenic diabetes insipidus (NDI) (Am J Hum Genet 69:738, 2001). The mutations were found in the C-terminus of AQP2 (721delG, 763 to 772del, and 812 to 818del). The wild-type AQP2 is a 271 amino acid protein, whereas these mutant genes were predicted to encode 330 to 333 amino acid proteins due to the frameshift mutations leading to the creation of a new stop codon 180 nucleotides downstream. The Xenopus oocyte expression study suggested that the trafficking of the mutant AQP2s was impaired. METHODS: To determine the cellular pathogenesis of these NDI-causing mutations in mammalian epithelial cells, Madin-Darby canine kidney (MDCK) cells were stably transfected with the wild-type AQP2, or the 763 to 772del mutant AQP2, or both. Cells were grown on the membrane support to examine the localization of AQP2 proteins by immunofluorescence microscopy. RESULTS: Confocal immunofluorescence microscopy showed that the wild-type AQP2 was expressed in the apical region, whereas the mutant AQP2 was apparently located at the basolateral region. Furthermore, the wild-type and mutant AQP2s were colocalized at the basolateral region when they were cotransfected, suggesting the formation of mixed oligomers and thereby mistargeting. CONCLUSION: Mixed oligomers of the wild-type and the 763 to 772del mutant AQP2s are mistargeted to the basolateral membrane due to the dominant-negative effect of the mutant. This defect is very likely to explain the pathogenesis of autosomal-dominant NDI. The mistargeting of the apical membrane protein to the basolateral membrane is a novel molecular pathogenesis of congenital NDI.

Animals↗
PubMed2003

Expression and function of the developmental gene Wnt-4 during experimental acute renal failure in rats.

The Wnt-beta-catenin pathway plays key roles in embryogenesis. Wnt-4 is known to be expressed in the mesonephric duct in embryonic development. It is tempting to speculate that the Wnt-4-beta-catenin pathway contributes to the recovery from acute renal failure (ARF). This study used an in vivo model of ARF rats to clarify the significance of the Wnt-4-beta-catenin pathway in ARF. ARF was induced by clamping the rat left renal artery for 1 h. At 3, 6, 12, 24, 48, and 72 h after reperfusion, whole kidney homogenate and total RNA were extracted for examination by Western blot analysis and real-time RT-PCR. Wnt-4 mRNA and protein expression were strongly increased at 3 to 12 h and 6 to 24 h after ischemia, respectively. In immunohistologic examination, Wnt-4 was expressed in the proximal tubules and co-expressed with aquaporin-1, GM130, and PCNA. Cyclin D1 and cyclin A were expressed at 24 to 48 h after reperfusion. In addition, the overexpression of Wnt-4 and beta-catenin promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Taken together, these data suggest that the Wnt-4-beta-catenin pathway plays a key role in the cell cycle progression of renal tubules in ARF. The Wnt-4-beta-catenin pathway may regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF.

Acute Kidney Injury↗
PubMed2002

Cloning and identification of a new member of water channel (AQP10) as an aquaglyceroporin.

Recently, a new member of aquaporins was reported as AQP10 [Biochem. Biophys. Res. Commun. 287 (2001) 814], which is incompletely spliced to lose the sixth transmembrane domain and has poor water and no glycerol/urea permeabilities. Independently, we identified a similar clone in human. Our AQP10 consists of 301 amino acids with a highly conserved sixth transmembrane domain. AQP10 has higher identity with aquaglyceroporins (50% with AQP9, 48% with AQP3, 42% with AQP7) and lower identity with other aquaporins (32% with AQP1 and AQP8). AQP10 is expressed only in the small intestine with (approximately 2 kb). RNase protection assay revealed the absence of the unspliced form, supporting the authenticity of our clone. When expressed in Xenopus oocytes, AQP10 stimulated osmotic water permeability sixfold in a mercury-sensitive manner. Glycerol and urea uptakes were also stimulated, while adenine uptake was not. The genome structure of AQP10 is similar to those of other aquaglyceroporins (AQP3, AQP7, AQP9) with six exons. We conclude that AQP10 represents a new member of aquaglyceroporins functionally as well as structurally.

Amino Acid Sequence↗
PubMed2002

Gene transfer of Smad7 using electroporation of adenovirus prevents renal fibrosis in post-obstructed kidney.

BACKGROUND: Unilateral ureteral obstruction (UUO) leads to interstitial fibrosis of the obstructed kidney, and TGF-beta is considered to play an important role in this fibrotic process. Smad7 has been recently identified as an antagonist of TGF-beta signaling. To investigate whether this novel molecule can be exploited for therapy of renal fibrosis, we determined the effect of exogenous Smad7, introduced by a recombinant adenovirus vector combined with in vivo electroporation (EP), on UUO-induced renal fibrosis in rats. METHODS: A model of UUO was made in SD rats by ligating their left ureters. The next day, the rats were divided into four groups and adenovirus was injected into the extended pelvic space (two groups received AdCMV-LacZ and two groups received AdCMV-Smad7). Then, EP was performed in one group of AdCMV-LacZ-injected rats and one group of AdCMV-Smad7-injected rats. The renal tissues were obtained 3, 5, 10, and 14 days after the UUO operation. We detected the efficiency of transgene by immunoblots of renal cortical and medullary tissues and immunohistochemical studies for Smad7 and FLAG (the FLAG gene was introduced in the AdCMV-Smad7 as a marker). The renal fibrosis was monitored by histological scoring of Masson stainings. RESULTS: In immunoblotting, both Smad7 and FLAG were clearly detected in the renal medullary tissue of the rats given AdCMV-Smad7 with EP. In contrast, immunoblots of renal cortical tissue did not demonstrate positive bands. In immunohistological study, Smad7 was stained in the renal medulla in the rats given AdCMV-Smad7 with EP. In the rats given AdCMV-Smad7 without EP, only a weak signal was detected in renal medullary tissue. The rats given AdCMV-Smad7 with EP demonstrated significantly more suppression of renal fibrosis than rats treated with AdCMV-LacZ. The rats treated with AdCMV-Smad7 without EP did not demonstrate significant suppression of renal fibrosis. CONCLUSION: These data indicate that gene transfer of Smad7 prevents UUO-induced renal fibrosis, suggesting that Smad7 may be applicable for the treatment of renal fibrosis. In vivo electroporation of adenovirus may be a powerful tool for gene delivery in renal tissue.

Adenoviridae↗
PubMed2002

Ligand-regulatable erythropoietin production by plasmid injection and in vivo electroporation.

BACKGROUND: The development of an in vivo gene transfer approach to deliver physiologic levels of recombinant proteins to the systemic circulation would represent a significant advance in the treatment of protein deficiency disorders. However, the ability to regulate transgene expression is of paramount importance for safe and effective gene transfer therapy. METHODS: We developed two plasmids, one encoder of chimeric GeneSwitch protein, and the other an inducible transgene for human erythropoietin (Epo). The level of secretion of Epo into the serum was modulated by intraperitoneal administration of mifepristone (MFP). Rats were divided into four groups: one group administered Epo plasmid with MFP for 50 days, a second group administered Epo plasmid with MFP for 15 days and then again from day 30 to day 50, a third group administered Epo plasmid without MFP, and a fourth group administered control plasmid. A pair of electrodes was inserted into the muscle of the right thigh, 100 mg of each plasmid was injected, and in vivo electroporation (8 pulses at 100 V for 50 msec) was performed. RESULTS: The presence of vector-derived Epo mRNA was detected by RT-PCR only in the Epo plasmid and MFP(+) groups. The hematocrit levels increased continuously, from the pre-injection level of 41.2% to 55.0% on day 30 and 53.8% on day 50 in the Epo plasmid and MFP(+) groups. In the MFP re-challenged group, the hematocrit levels rose up to day 15, fell after 20 to 30 days, and then rose again after MFP re-administration. The serum Epo levels increased only in the Epo plasmid and MFP(+) groups. There were no significant changes in hematocrit levels and Epo levels in the Epo plasmid and MFP(-) group. CONCLUSION: Epo gene transfer with the GeneSwitch system by in vivo electroporation is a useful procedure for efficient drug-regulated delivery of Epo.

Animals↗