Rapamycin mediated caspase 9 homodimerization to safeguard human pluripotent stem cell therapy / 生物工程学报

Human induced pluripotent stem cells (hiPSCs) are promising in regenerative medicine. However, the pluripotent stem cells (PSCs) may form clumps of cancerous tissue, which is a major safety concern in PSCs therapies. Rapamycin is a safe and widely used immunosuppressive pharmaceutical that acts through heterodimerization of the FKBP12 and FRB fragment. Here, we aimed to insert a rapamycin inducible caspase 9 (riC9) gene in a safe harbor AAVS1 site to safeguard hiPSCs therapy by drug induced homodimerization. The donor vector containing an EF1α promoter, a FRB-FKBP-Caspase 9 (CARD domain) fusion protein and a puromycin resistant gene was constructed and co-transfected with sgRNA/Cas9 vector into hiPSCs. After one to two weeks screening with puromycin, single clones were collected for genotype and phenotype analysis. Finally, rapamycin was used to induce the homodimerization of caspase 9 to activate the apoptosis of the engineered cells. After transfection of hiPSCs followed by puromycin screening, five cell clones were collected. Genome amplification and sequencing showed that the donor DNA has been precisely knocked out at the endogenous AAVS1 site. The engineered hiPSCs showed normal pluripotency and proliferative capacity. Rapamycin induced caspase 9 activation, which led to the apoptosis of all engineered hiPSCs and its differentiated cells with different sensitivity to drugs. In conclusion, we generated a rapamycin-controllable hiPSCs survival by homodimerization of caspase 9 to turn on cell apoptosis. It provides a new strategy to guarantee the safety of the hiPSCs therapy.

Puromycin aminonucleoside triggers apoptosis in podocytes by inducing endoplasmic reticulum stress

BACKGROUND: Puromycin aminonucleoside (PAN) is a known podocytotoxin. PAN-induced nephrosis is a widely used animal model for studying human idiopathic nephrotic syndrome. Abnormal protein accumulation associated with podocyte-specific endoplasmic reticulum (ER) stress damages cells structurally and functionally, which in turn induces apoptosis and severe proteinuria. In the present study, we investigated the effect of PAN on ER stress and apoptosis in podocytes in vitro. METHODS: Mouse podocytes were cultured and treated with various concentrations of PAN. ER stress markers were then evaluated by western blotting, and apoptosis was evaluated by fluorescence-activated cell sorting (FACS) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. RESULTS: PAN treatment increased ER stress markers such as activating transcription factor (ATF) 6α and caspase-12 in a dose-dependent manner at 12 and 24 hours, respectively. These markers were reduced by chemical chaperones, such as sodium 4-phenylbutyric acid and tauroursodeoxycholic acid. PAN treatment also increased 78 kD glucose-regulated protein (GRP78)/binding immunoglobulin protein (BiP) at the earlier stage of 12 hours. PAN significantly induced podocyte apoptosis in concentration- and time-dependent manners, as seen using FACS and TUNEL assays. This result was improved by Nox4 siRNA, ATF6 siRNA, and chemical chaperones. LY294002, a PI3-kinase inhibitor, significantly boosted ER stress and apoptosis. PAN-induced ER stress increased oxidative stress and subsequently induced apoptosis, and could be mitigated by inhibition of PI3-kinase signaling. CONCLUSION: Our findings suggest that PAN induces ER stress in podocytes mainly through the GRP78/BiP, ATF6α, and caspase-12 pathways, which trigger apoptosis via induction of oxidative stress. This stress is mitigated by inhibiting PI3-kinase signaling.

High prevalence of methicillin resistance and PVL genes among Staphylococcus aureus isolates from the nares and skin lesions of pediatric patients with atopic dermatitis

Staphylococcus aureus is highly prevalent among patients with atopic dermatitis (AD), and this pathogen may trigger and aggravate AD lesions. The aim of this study was to determine the prevalence of S. aureus in the nares of pediatric subjects and verify the phenotypic and molecular characteristics of the isolates in pediatric patients with AD. Isolates were tested for antimicrobial susceptibility, SCCmec typing, and Panton-Valentine Leukocidin (PVL) genes. Lineages were determined by pulsed-field gel electrophoresis and multilocus sequence typing (MLST). AD severity was assessed with the Scoring Atopic Dermatitis (SCORAD) index. Among 106 patients, 90 (85%) presented S. aureus isolates in their nares, and 8 also presented the pathogen in their skin infections. Two patients had two positive lesions, making a total of 10 S. aureus isolates from skin infections. Methicillin-resistant S. aureus (MRSA) was detected in 24 (26.6%) patients, and PVL genes were identified in 21 (23.3%), including 6 (75%) of the 8 patients with skin lesions but mainly in patients with severe and moderate SCORAD values (P=0.0095). All 24 MRSA isolates were susceptible to trimethoprim/sulfamethoxazole, while 8 isolates had a minimum inhibitory concentration (MIC) to mupirocin >1024 μg/mL. High lineage diversity was found among the isolates including USA1100/ST30, USA400/ST1, USA800/ST5, ST83, ST188, ST718, ST1635, and ST2791. There was a high prevalence of MRSA and PVL genes among the isolates recovered in this study. PVL genes were found mostly among patients with severe and moderate SCORAD values. These findings can help clinicians improve the therapies and strategies for the management of pediatric patients with AD.

Effect of Puromycin Aminonucleoside on Podocyte P-Cadherin

PURPOSE: To test whether the expression of P-cadherin, a component of slit diaphragms between podocyte foot processes, would be altered by puromycin aminonucleoside (PAN) in a cultured podocyte in vitro. METHODS: Rat glomerular epithelial cells (GEpC) were cultured with various concentrations of PAN. The distribution of P-cadherin was examined with a confocal microscope. Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to measure the change in P-cadherin expression. RESULTS: This study found that P-cadherin was concentrated in the inner and peripheral cytoplasm with high concentrations of PAN under immunofluorescence views. Western blotting of GEpC revealed that PAN induced a decrease of P-cadherin in dose- and time-dependent manners. A high dose (50 microg/mL) of PAN decreased P-cadherin expression by 21.9% at 24 h (P<0.05) and 31.9% at 48 h (P<0.01) compared to those without PAN. In RT-PCR, high concentrations (50 microg/mL) of PAN also decreased P-cadherin mRNA expression, similar to protein suppression, by 23.5% at 48 h (P<0.05). CONCLUSION: Podocytes exposed to PAN in vitro concentrated P-cadherin internally, and reduced P-cadherin mRNA and protein expression. This could explain the development of proteinuria in experimental PAN-induced nephropathy.

Puromycin aminonucleoside modulates p130Cas of podocytes / 소아과

PURPOSE: Puromycin aminonucleoside (PAN) specifically injures podocytes, leading to foot process effacement, actin cytoskeleton disorganization, and abnormal distribution of slit diaphragm proteins. p130Cas is a docking protein connecting F-actin fibers to the glomerular basement membrane (GBM) and adapter proteins in glomerular epithelial cells (GEpCs; podocytes). We investigated the changes in the p130Cas expression level in the PAN-induced pathological changes of podocytes in vitro. METHODS: We observed changes in the p130Cas expression in cultured rat GEpCs and mouse podocytes treated with various concentrations of PAN and antioxidants, including probucol, epigallocatechin gallate (EGCG), and vitamin C. The changes in the p130Cas expression level were analyzed using confocal immunofluorescence imaging, Western blotting, and polymerase chain reaction. RESULTS: In the immunofluorescence study, p130Cas showed a diffuse cytoplasmic distribution with accumulation at distinct sites visible as short stripes and colocalized with P-cadherin. The fluorescences of the p130Cas protein were internalized and became granular by PAN administration in a dose-dependent manner, which had been restored by antioxidants, EGCG and vitamin C. PAN also decreased the protein and mRNA expression levels of p130Cas at high doses and in a longer exposed duration, which had been also reversed by antioxidants. CONCLUSION: These findings suggest that PAN modulates the quantitative and distributional changes of podocyte p130Cas through oxidative stress resulting in podocyte dysfunction.

The Change of Podocyte beta-Catenin by Puromycin Aminonucleoside

PURPOSE: To test whether the expression of beta-catenin, a component of podocyte as a filtration molecule, would be altered by puromycin aminonucleoside (PAN) in the cultured podocyte in vitro. METHODS: We cultured rat glomerular epithelial cells (GEpC) with various concentrations of PAN and examined the distribution of beta-catenin by confocal microscope and measured the change of beta-catenin expression by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: We found that beta-catenin relocalized from peripheral cytoplasm to inner cytoplasm, therefore, intercellular separations were seen in confluently cultured cells by high concentrations of PAN in immunofluorescence views. In Western blotting of GEpC, PAN (50 microg/mL) decreased beta-catenin expression by 34.9% at 24 hrs and 34.3% at 48 hrs, compared to those in without PAN condition (P<0.05). In RT-PCR, high concentrations (50 microg/mL) of PAN also decreased beta-catenin mRNA expression similar to protein suppression by 25.4% at 24 hrs and 51.8% at 48 hrs (P<0.05). CONCLUSION: Exposure of podocytes to PAN in vitro relocates beta-catenin internally and reduces beta-catenin mRNA and protein expression, which could explain the development of proteinuria in experimental PAN-induced nephropathy.

Effects of Dietary Salt Restriction on Puromycin Aminonucleoside Nephrosis: Preliminary Data

Proteinuria is a major promoter that induces tubulointerstitial injury in glomerulopathy. Dietary salt restriction may reduce proteinuria, although the mechanism is not clear. We investigated the effects of dietary salt restriction on rat kidneys in an animal model of glomerular proteinuria. Male Sprague-Dawley rats were used and divided into 3 groups: vehicle-treated normal-salt controls, puromycin aminonucleoside (PA)-treated normal-salt rats, and PA-treated low-salt rats. PA was given at a dose of 150 mg/kg BW at time 0, followed by 50 mg/kg BW on days 28, 35, and 42. Sodium-deficient rodent diet with and without additional NaCl (0.5%) were provided for normal-salt rats and low-salt rats, respectively. On day 63, kidneys were harvested for histopathologic examination and immunohistochemistry. PA treatment produced overt proteinuria and renal damage. Dietary salt restriction insignificantly reduced proteinuria in PA-treated rats, and PA-treated low-salt rats had lower urine output and lower creatinine clearance than vehicle-treated normal-salt controls. When tubulointerstitial injury was semiquantitatively evaluated, it had a positive correlation with proteinuria. The tubulointerstitial injury score was significantly increased by PA treatment and relieved by low-salt diet. ED1-positive infiltrating cells and immunostaining for interstitial collagen III were significantly increased by PA treatment. These changes appeared to be less common in PA-treated low-salt rats, although the differences in PA-treated normal-salt versus low-salt rats did not reach statistical significance. Our results suggest that renal histopathology in PA nephrosis may potentially be improved by dietary salt restriction. Non-hemodynamic mechanisms induced by low-sodium diet might contribute to renoprotection.

Changes in Endothelin Receptor Type B and Neuronal Nitric Oxide Synthase in Puromycin Aminonucleoside-Induced Nephrotic Syndrome

The collecting duct endothelin (ET) system, which involves ET-1 and its two receptors, may play a role in the regulation of renal sodium in association with the nitric oxide synthase (NOS) system. We determined whether sodium retention is associated with changes in the endothelin and NOS systems at different stages (i.e., a sodium retaining stage and a compensatory stage) of nephrotic syndromes. On day 7 after puromycin aminonucleoside (PAN) injection, urinary sodium excretion was decreased, ascites had developed, and there was a positive sodium balance. ET-1 mRNA expression was increased in the inner medulla of the kidney, whereas protein expression of ET receptor type B (ET(B)R) was unchanged. The expression of neuronal NOS (nNOS) was decreased in the inner medulla. On day 14, urinary sodium excretion was unchanged compared with controls. The expression of ET(B)R increased, while nNOS expression in the inner medulla was comparable to controls. These findings suggest that decreased nNOS plays a role in the development of sodium retention in the nephrotic syndrome. Recovery of nNOS and increased renal ET(B)R synthesis may promote sodium excretion in later stages of the nephrotic syndrome (on day 14).

Changes of Atrial Natriuretic Peptide System in Rats with Puromycin Aminonucleoside-Induced Nephrotic Syndrome

Sodium retention is a hallmark of nephrotic syndrome. We investigated whether sodium retention is associated with changes of natriuretic peptide system at different stages (i.e., a sodium retaining stage and a compensatory stage) of nephrotic syndrome. At day 7 after PAN (puromycin aminonucleoside) injection, the urinary excretion of sodium was decreased, along with the development of ascites and positive sodium balance. The plasma and urinary ANP (atrial natriuretic peptide) immunoreactivities were increased. ANP mRNA expression was increased in the heart and kidney, whereas that of NPR (natriuretic peptide receptor)-A and NPR-C mRNA was decreased in the kidney. The expression of NEP was decreased in the kidney. At day 14, urinary excretion of sodium did not differ from the control. The plasma ANP level and heart ANP mRNA expression returned to their control values. The expression of ANP mRNA in the kidney was increased in association with increased urinary ANP immunoreactivities. The expression of NPR-A in the kidney became normal, whereas that of NPR-C kept decreased. The expression of NEP (neutral endopeptidase) remained decreased. These findings suggest that the increased renal ANP synthesis in association with decreased metabolism via NEP and NPR-C may play a compensatory role against the development of sodium retention in nephrotic syndrome. The decreased of NPR-A expression in the kidney may contribute to the ANP resistance at day 7. The subsequent recovery of NPR-A expression may play a role in promoting sodium excretion in later stage (at day 14).

Effects of puromycin aminonucleoside on the cytoskeletal changes of glomerular epithelial cells / 소아과

PURPOSE:This study was designed to clarify the mechanism of proteinuria in nephrotic syndrome patients by using puromycin aminonucleoside (PAN) nephrosis model. METHODS:Following administration of various concentrations of PAN and antioxidants we observed the changes of podocyte cytoskeletons in cultured rat glomerular epithelial cells (GEpC) by method of scanning electron microscope, reactive oxyten species (ROS) analysis, permeability assay, confocal microscope, and Western blot assay. RESULTS:PAN not only induced the ultrastructural changes of GEpC, such as shortening and fusion of microvilli, but also separated the intercellular gaps and linear ZO-1. PAN induced oxidative stresses in time and dose dependent manners and increases of intercellular permeability in anti-oxidants inhibitable manners. High concentration of PAN induced not only actin polymerization and disorganization, but also the conglomerulation and internal dislocation of alpha-actinin protein. The intensities of fluorescences of ZO-1 protein were diminished and internalized by PAN in a dose-dependent manner, which were inhibited by anti anti-oxidants. CONCLUSION:PAN induced the changes of podocytes cytoskeleton and junctional barriers by way of increasing ROS in GEpC that resulted in increasing their permeability in a antioxidatn-inhibitable manner. Glomerular hyperpermeability induced by PAN mediateing through oxidative stresses is thought to take part in the mechanism of proteinuria in nephrotic syndrome. (Korean J Pediatr 2008;51:54-61)

Construction of recombinant retroviral vector carrying Lab gene of foot-and-mouth disease virus and its expression in bovine kidney (MDBK) cells / 生物工程学报

In this study, foot-and-mouth disease virus (FMDV) strain OA/58 RNAs were used as templates for RT-PCR. By the molecular cloning, the Lab gene encoding leader protease called Lpro were cloned in retroviral vector pBPSTR1 to obtain reconstruction retroviral vector termed pBPSTR1-Lab. At different concentrations of puromycin and tetracycline respectively in the cell culture mediums, the growth of bovine kidney cells (MDBK) showed that the optimal puromycin resistant selection concentration was 3 microg/mL and tetracycline regulatory concentration was 1 microg/mL. Pseudotyped retroviral virus particles were produced by transiently co-tansfecting GP2-293 cells with a retroviral vector DNA and VSV-G plasmid. Then MDBK cells were infected by pseudotyped retroviral virus and were continually seeded in the medium at the optimal tetracycline regulatory concentration and puromycin selection concentration for 12 days to obtain puromycin resistant colonies whose genomes contained the Lab gene. After tetracycline removal, synthesis of Lpro induced severe morphological changes in the puromycin resistant MDBK cells. PCR and Western blotting proved that a stable MDBK cell line inducibly expressing the Lab gene under the control of tetracycline was obtained. The experiment might provide a basis for studying that Lpro of FMDV plays an important role in MDBK cell pathogenesis.

Effect of Glomerular Cyclooxygenase-2 Overexpression on Podocyte Injury Induced by Puromycin in Mice / 대한신장학회지

PURPOSE: The aim of this study is to investigate the effect of glomerular cyclooxygenase-2 (COX-2) overexpression on podocyte injury by puromycin aminonucleoside (PAN) in nephrin-driven COX-2 transgenic mice. METHODS: We administrated PAN intravenously on day-1 (15 mg/100 g body weight) and day-3 (30 mg/100 g body weight) in wild type (male B6/D2 mice) and COX-2 transgenic mice. An additional group received a selective COX-2 inhibitor (SC58236) with PAN. The animals from each group were sacrificed at the end of day-3 and 10. We investigated albuminuria, foot process effacement and glomerular COX-2 and nephrin expression. RESULTS: PAN induced albuminuria only in COX-2 transgenic mice, with the peak on day-3. Selective COX-2 inhibition significantly reduced albuminuria. EM examination demonstrated foot process effacement, which was improved partially by selective COX-2 inhibition, in COX-2 transgenic mice treated with PAN on day-3. Glomerular COX-2 expression increased significantly on day-3 in COX-2 transgenic mice treated with PAN, whereas expression of nephrin showed a tendency to decrease on day-3. These changes of expression of COX-2 and nephrin were partly attenuated by selective COX- 2 inhibition. Unlike COX-2 transgenic mice, wild-type mice did not show any changes even after PAN treatment. CONCLUSION: In COX-2 transgenic mice, PAN induced albuminuria, podocyte injury and up-regulation of glomerular COX-2, which were ameliorated by selective COX-2 inhibitor. These results suggest that COX-2 may play an important role in increasing susceptibility of podocytes to injury and selective COX-2 inhibition may ameliorate podocyte injury.

Overexpression of the Fanconi Anemia A Gene in Hela and MCF10A Cells

BACKGROUND: Fanconi Anemia (FA) is an autosomal recessive inherited disease, which is characterized by developmental abnormalities, progressive bone marrow failure and a predisposition to cancer. The phenotypes of FA cells show extreme sensitivities towards oxygen and DNA cross linking agents, such as diepoxybutane and mitomycin C (MMC). METHODS: In the current study, retroviruses expressing the FANCA gene were prepared to create the stable cell lines, Hela (cervical carcinoma) and MCF10A (breast). The expression of FANCA protein in the Hela and MCF10A stable cells, following puromycin selection, was checked using Western blot. The difference in the cell growth between the parent and FANCA expressing cells following MMC treatment was checked using the MTT assay. RESULTS: The expression of exogenous FANCA protein in the Hela and MCF10A stable cells was observed using Western blot. The MCF10A cells expressing exogenous FANCA were resistant to MMC concentrations with the range 0.01~1 micrometer compared with the MCF10 parent cells. However, at an MMC concentration of 10 micrometer, there was no difference in the susceptibility between the parent and FANCA expressing MCF10 cells. The Hela cells expressing FANCA showed no resistance at any MMC concentration (0.01~10 micrometer). CONCLUSION: FANCA protein is an important factor for resistance to the cross linking agent, MMC, in MCF10A breast cells, but not in Hela cervical carcinoma cells.

Sex chromosome analysis and IGF-II expression on activated human unfertilized oocytes after ICSI with calcium ionophore A23187 and puromycin / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the sex chromosomes and the expression of insulin-like growth factor-II (IGF-II) on activated human unfertilized oocytes after intracytoplasmic sperm injection(ICSI) with calcium ionophore A23187 and puromycin.</p><p><b>METHODS</b>All 95 discarded oocyes that showed no evidence of fertilization at 16-18 h after in vitro maturation and intracytoplasmic sperm injection cycles (IVM-ICSI)/conventional ICSI were exposed to calcium ionophore A23187 (5 micromol/L) for 5 min and then were incubated with puromycin (10 microg/mL) for 4 h. After activation, the oocytes were cultured in vitro for 3-5 days. The sex chromosome analysis was performed by dual color fluorescence in situ hybridization. The expression of IGF-II on the activated embryos, normal embryos, and parthenotes was examined.</p><p><b>RESULTS</b>The combination of calcium ionophore A23187 with puromycin could activate the unfertilized oocytes 22 h after ICSI. The activated rate, cleavage rate, and quality of activated embryos of the IVM-ICSI group were similar to those of ICSI group, respectively. Sex chromosome analysis indicated that 8 male and 5 female embryos had been derived from two pronucleus and a second polar body. The expression of IGF-II on activated embryos and normal embryos was high and similar, which was much stronger than that of parthenotes.</p><p><b>CONCLUSION</b>The combination of calcium ionophore A23187 with puromycin could effectively activate unfertilized oocytes 22 h after ICSI. Moreover, the unfertilized oocytes activated by calcium ionophore A23187 and puromycin had normal sex chromosomes and expression of IGF-II like the normal embryos. These suggest that oocyte activation may be considered as a remedial measure in the presence of total or nearly total fertilization failure in ICSI.</p>

HygR and PurR plasmid vectors for episomal transfection of Trypanosoma cruzi

This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi.

Effects of steroid hormones on total brain Na(+)-k+ ATPase activity in Oreochromis mossambicus.

The effects of administration of cortisol, corticosterone, testosterone, progesterone and a synthetic estrogen. diethylstilbestrol (DES) on total brain Na(+)-K+- ATPase were investigated in tilapia, O. mossambicus. Exogenous administration of 0.125 and 0.25 microg/g body weight of glucocorticoids and 0.125, 0.25 and 0.5 microg/g body weight of DES for 5 days significantly stimulated Na+(-) K+ ATPase activity by 14-41% in the brain, while 0.5 microg/g body weight of glucocorticoids did not evoke any response on the activity of the enzyme. Progesterone (0.125 and 0.25 microg/g body weight) administration significantly decreased the enzyme activity by 21-36% and high dose (0.5 microg/g body weight) was ineffective. Testosterone exhibited a biphasic effect on Na(+)-K+ ATPase activity--a low dose stimulated by 14% while middle and high doses inhibited it by 19-24%. The results seem to be the first report on the effect of steroids on brain ATPase activity in a teleost. When 0.25microg/g body weight of actinomycin D or puromycin was administered prior to the treatment of similar doses of hormones, the inhibitors significantly inhibited the effect of the hormones by 24-52%. This clearly shows that the effect of the hormones was sensitive to the action of inhibitors suggesting a possible genomic mode of action under long-term treatment. The results suggest that cortisol, corticosterone and DES may possibly stimulate the co-transport of glucose and excitation of membrane potential while progesterone and testosterone inhibit them in the brain of O. mossambicus by regulating the activity of Na(+)-K+ ATPase.

The Effects of Selective Cyclooxygenase-2 Inhibition in Puromycin Aminonucleoside Nephropathy Rats / 대한신장학회잡지

BACKGROUND: It is known that non-steroidal antiinflammatory drugs (NSAIDs) reduce the amount of proteinuria in nephrotic syndrome. It is based on the facts that the NSAIDs block the production of prostaglandins. Therefore selective cyclooxygenase-2 (COX-2) inhibitor may be expected to play a role in reduction of the proteinuria in nephrotic syndrome. METHODS: Twenty-seven Sprague-Dawley rats were divided into 3 groups. After 3 to 5 days of adaptation, we gave puromycin aminonucleoside to groups A and B via intraperitoneal route. The third group C was a normal control group. Selective COX-2 inhibitor was orally given to group A for 2 weeks. Each group was divided again into 3 subgroups by the day of experiment: 1, 14 and 21-day subgroups. We checked the changes in the serum and urine creatinine, albumin concentrations, creatinine clearances, the amount of proteinuria and the pathologic findings. The differences between groups were tested by 2-way ANOVA and Dunnett T-test, and the changes of proteinuria were tested by Repeated measures ANOVA. RESULTS: The changes of 24-hour urine protein excretion were significantly different between three groups (p<0.01). Protein excretion of group A was significantly decreased, especially between 14 and 21 days (p<0.05). The changes of creatinine clearance were significantly different between three groups (p<0.05), between 1 and 21 days (p<0.05). Electron microscopy showed morphological recovery of foot processes after administration of selective COX-2 inhibitor in PAN nephropathy rats (group A). CONCLUSION: It is suggested that selective COX- 2 inhibitors may be effective in reducing proteinuria and protecting the renal function in nephrotic syndrome.

The Effect of Atorvastatin on the Development of Puromycin Aminonucleoside(PAN)-induced Nephrosis in Rats

PURPOSE: Several studies have suggested that hyperlipidemia might be a causative factor contributing to the progression of initial glomerular injury through the development of glomerulosclerosis. We examined the potential beneficial effect of atorvastatin - which blocks the rate limiting step of cholesterol synthesis by inhibiting HMG-CoA reductase - in PAN- induced nephrosis. MATERIALS AND METHODS: Glomerulosclerosis was induced in Sprague-Dawley male rats by repeated administration of PAN. Sprague-Dawley male rats were divided into 3 groups:group I(control), group II(PAN 20 mg/kg, subcutaneous injection), group III(PAN 20 mg/kg subcutaneous injection and atorvastatin 50 mg/kg/day per oral). On the 11th week, upon sacrifice of the experimental animals, blood sampling, 24-hr urine collection and nephrectomy were performed. RESULTS: Group III had significantly lower BUN and higher serum albumin(30.9+/-17.2 vs. 17.3+/-2.5 mg/dL; 2.3+/-0.1 vs. 2.5+/-0.2 g/dL, P0.05) compared with group II. Atorvastatin administration lowered the glomerular sclerosing index significantly(26.2% vs. 13.3%, P<0.05). CONCLUSION: Puromycin-induced glomerulosclerosis could be ameliorated by the reduction of hyperlipidemia with atorvastatin. This suggests that hyperlipidemia contributes to the pathogenesis of glomerulosclerosis.

Altered Expression of Renal AQP1, AQP2 and NHE3 in Puromycin Aminonucleoside Induced Nephrotic Syndrome: Intervention by Alpha-MSH Treatment / 대한신장학회잡지

BACKGROUND: We examined whether puromycin aminonucleoside (PAN)-induced nephrotic syndrome (NS) is associated with altered renal handling of water and sodium along with changes of renal abundance of aquaporins (AQP1 and AQP2) and NHE3. Next we tested the effects of alpha-melanocyte stimulating hormone (alpha- MSH), a potent anti-inflammatory drug, on the PAN-induced renal functional derangement and the changes of renal AQPs and NHE3 abundance. METHODS: PAN was administered to Sprague-Dawley rats using two protocols: protocol 1 (180 mg/kg, single iv injection) and protocol 2 (100 mg/kg, single iv injection). RESULTS: In both protocols, PAN-induced NS was associated with decreased urine concentration, manifested by an increased urine output and decreased urine osmolality and TcH2O. Consistent with this, a marked downregulation of vasopressin-regulated collecting duct AQP2 expression was seen in PAN-induced NS. In protocol 2 where rats treated with moderate dose of PAN, alpha-MSH cotreatment prevented the reduction of urine osmolality and the increase of the FENa in the PAN-induced NS. This suggests that alpha-MSH may have protective effects against the renal functional deterioration induced by PAN. The renal abundance of the AQP1, AQP2 and NHE3 was reduced in PAN-induced NS in protocol 2, as seen in protocol 1. In contrast to the functional improvement, alpha-MSH cotreatment had marginal effects in the prevention of renal AQP1, AQP2 and NHE3 downregulation in PAN-induced NS. CONCLUSION: PAN-induced NS was associated with decreased urine concentration along with reduced renal AQP2, AQP1 and NHE3 abundance. Alpha-MSH may have protective effects against the renal functional deterioration (e.g., urine osmolality and FENa). However, alpha-MSH treatment alone is less likely to prevent the marked reduction of AQP2, AQP1 and NHE3 abundance in PAN-induced NS, in contrast to the previously known dramatic effects against the ischemia-reperfusion injury in kidney and small intestine.

Isolation and Complete Nucleotide Analysis of a Noncytopathic BVDV Variant using a Dominant Selective Marker

Upon infection, bovine viral diarrhea virus (BVDV), one of the pestiviruses in the Flaviviridae family, is divided into two biotypes, cytopathic (cp) and noncytopathic (ncp). The mechanism of cytopathogenicity, however, is not elucidated yet. In this study, we have investigated viral genetic element affecting the cytopathogenicity of BVDV by using an infectious cDNA molecular clone containing a dominant selective marker, puromycin acetyltransferase gene (pNADL/pac). From the recombinant cDNA clone pNADL/pac, viral RNA was synthesized by T7 RNA polymerase in vitro. By selecting the MDBK cells transfected with in vitro transcribed cp NADL/pac viral RNA with puromycin, we obtained the selected MDBK cells harboring cp NADL/pac viral RNA, which did not show cytopathic effect and could be passaged. Little cytopathic effect was observed upon infection of naive MDBK cells with the viral particles released from the selected cp NADL/pac-transfected MDBK cells. Complete nucleotide analysis of viral particles released from cp NADL/pac-selected MDBK cells revealed 5 substitutions in the viral open reading frame, but not in the 5' and 3'NTRs. Interestingly, only two point mutations (G8147A and T11343C) changed their amino acid code (M2588V in NS4B and T2653M in NSSB), and the other 3 substitutions (C1627T, T3097C, and T3376C) was silent. Therefore, our results suggest that NS4B and NSSB proteins of BVDV may play a role in cytopathogenicity.