ANTIPROTEINURIC EFFECT OF CALCIUM ANTAGONISTS ON PUROMYCIN-INDUCED EXPERIMENTAL NEPHROSIS

Calcium antagonists have a potential for beneficial effects on kidney function unrelated to their antihypertensive action. In this study we have investigated the efficacy of calcium antagonists compounds (verapamil, nifedipine and diltiazem) on reversible acute renal insufficiency, proteinuria and interstitial nephritis induced by the puromycin ammo nucleoside (PAN). An increase in blood pressure (BP) was detected on day 14, with no statistical differences in the response to calcium antagonists. Serum creatinine concentration increased to 1.2 mg/dL on day 7 after PAN and decreased to 0.7 mg/dL at 14 days, calcium antagonists shortened the time required to reach baseline or control levels. Calcium antagonists also reduced proteinuria in the PAN-treated animals, in both day 7 and day 14. Differential effects of the antagonists were observed. Verapamil caused a greater reduction (p < 0.01) in proteinuria than nifedipine or diltiazem in day 7. Moreover, verapamil (p < 0.01) and nifedipine (p < 0.01) reduced the total number of interstitial infiltrating leukocytes from 690 to 120 and 425 positive cells/20 high power fields (× 63) respectively, by contrast, diltiazem had no effect. We conclude that in this model of PAN nephropathy verapamil is more effective in reducing both proteinuria and the severity of acute interstitial nephritis than either nifedipine or diltiazem. The possible clinical implications of these results remain to be elucidated.     

锰苯甲酸卟啉在嘌呤霉素氨基核苷诱导的大鼠肾脏病模型中的作用

目的:探讨超氧化物歧化酶类似物锰苯甲酸卟啉(MnTBAP)在嘌呤霉素氨基核苷(puromycin aminonucleoside,PAN)诱导的大鼠肾脏病模型中的作用?方法:24只雄性SD大鼠按照每组8只随机分为正常对照组?模型组(一次性尾静脉注射150 mg/kg PAN建立PAN诱导的大鼠肾脏病模型)?MnTBAP治疗组[造模大鼠于造模当天开始腹腔注射MnTBAP 10 mg/(kg·d),共给药14 d]?收集大鼠24 h尿液,Bradford法检测24 h尿蛋白总量;应用透射电镜观察肾小球足细胞的超微结构;实时荧光定量PCR法在mRNA水平检测足细胞裂孔隔膜蛋白Nephrin和Podocin的表达量;Western blot在蛋白质水平检测Nephrin和Podocin的表达量?结果:①与正常对照组相比,模型组大鼠的24 h尿蛋白总量明显升高(P < 0.01),肾脏指数升高(P < 0.01);MnTBAP治疗后大鼠24 h尿蛋白总量较模型组下降(P < 0.01),肾脏指数较模型组下降(P < 0.01);②透射电镜观察结果表明,模型组大鼠的肾小球足细胞足突出现广泛融合甚至消失,MnTBAP治疗后这种现象得以改善;③实时荧光定量PCR结果显示,模型组大鼠肾皮质Nephrin和Podocin的mRNA表达较正常对照组降低(P < 0.01),MnTBAP治疗组Nephrin和Podocin的mRNA表达较模型组增加(P < 0.01);④Western blot结果显示,模型组大鼠肾皮质Nephrin和Podocin蛋白的表达较正常对照组降低(P < 0.01),MnTBAP治疗组Nephrin和Podocin蛋白的表达较模型组增加(P < 0.01)?结论:MnTBAP可以降低PAN诱导的肾病大鼠的蛋白尿,并明显减轻足细胞损伤?     

DMSO potentiates aminonucleoside of puromycin nephrosis in rats

Dimethyl sulphoxide (DMSO), 3 g/kg body weight, administered daily by the intraperitoneal route, potentiated the proteinuria and formation of tubular casts in aminonucleoside of puromycin (PA) induced nephrosis in Sprague-Dawley rats. The effect was evident at 4 as well as 8-9 days following PA administration. In the absence of PA, DMSO did not induce proteinuria or cast formation. The mechanism by which DMSO enhanced proteinuria and cast formation is not known.     

Tissue distribution of alpha-tocopherol in nephrotic rats

1. Reactive oxygen species are involved in the pathogenesis of puromycin aminonucleoside (PAN) nephrosis and alpha-tocopherol is one of the major anti-oxidants in the body. 2. In the present study, we measured the levels of alpha-tocopherol by high-performance liquid chromatography in the plasma and in nine tissues of control and nephrotic rats obtained 10 days after either 0.9% saline solution or PAN injection, respectively. 3. In nephrotic rats, alpha-tocopherol levels increased four-fold in plasma; however, the molar ratio of alpha-tocopherol/ cholesterol remained unchanged, suggesting that the increase in alpha-tocopherol content was attributable to an increase in plasma lipid concentration. 4. In nephrotic rats, the alpha-tocopherol/cholesterol ratio increased 1.33-fold in adrenal glands and 1.34-fold in the testis, but remained unchanged in heart, spleen, liver, kidney lung, brain and muscle. 5. These data suggest that, in PAN nephrotic rats, there are alterations in the distribution of alpha-tocopherol and there is no deficiency of alpha-tocopherol in plasma or tissues.     

Experimentally induced parthenogenetic activation of human oocytes

A total of 486 unfertilized, aged human oocytes were exposedto ethanol, calcium ionophore A23187, phorbol ester or puromycinand examined for evidence of activation. Five per cent of controloocytes (3/58) were spontaneously activated. Of the two agentswhich cause the release of intracellular Ca²⁺ ions, Ca²⁺ ionophoreinduced activation of only 16% of unfertilized oocytes, whileethanol did not have any effect. Phorbol ester, a stimulatorof protein kinase C, also resulted in limited activation (14%of oocytes). In contrast, puromycin, an inhibitor of proteinsynthesis, resulted in activation of 91% of the exposed oocytes.It is speculated that puromycin probably inhibits a specificcytostatic factor or factors which are responsible for maintenanceof the metaphase II block. Morphologically activated oocytesusually retained the second polar body and formed subnuclei.The developmental potential of activated oocytes appeared tobe reduced, with only some oocytes capable of a single division.     

Pregnancy following chemical activation of oocytes in a couple with repeated failure of fertilization using ICSI: case report

We report our attempts to achieve a successful pregnancy outcome with calcium ionophore A23187 and puromycin oocyte activation using sperm from a normozoospermic husband of a patient with previous repeated failed fertilization following ICSI. Oocytes from the female partner of a couple with a 4 year history of unexplained primary infertility with repeated failed fertilization following ICSI were used. In the latest ICSI attempt, oocytes were activated by treatment with calcium ionophore (5 min) and puromycin (5 h), then cultured. In this cycle, assisted oocyte activation with calcium ionophore and puromycin after ICSI resulted in a satisfactory fertilization rate (8/12; 66.7%); in prior cycles only one of 71 oocytes (1.4%) was fertilized. The outcome was a Caesarean section delivery of a healthy male infant without congenital abnormalities at 41 weeks, 2 days of gestation. In conclusion, the use of calcium ionophore and puromycin for oocyte activation was found to be a useful method in a case of repeated failed fertilization after ICSI.     

Late changes in the course of mitosis in a synchronized Chinese hamster cell culture after inhibition of RNA and protein synthesis

The effect of inhibition of synthesis of various types of RNA and proteins at different periods of interphase on the course of late mitosis was studied in a synchronized culture of Chinese hamster cells. Analysis of the mitotic index and forms of pathology of division showed that the action of different doses of actinomycin D and puromycin in the first half of interphase produces an identical effect: C-mitoses in the immediate and late waves of cell division. Suppression of synthesis of total cell RNA and proteins in the second half of interphase was accompanied by delay of the cells in metaphase, with scattering of the chromosomes, evidence of a disturbance of the synthesis of the components of the division spindle. It is suggested that proteins (tubulins) and RNA participate as a reserve pool in the organization of the division spindle of late mitosis.Laboratory of Cytology, Institute of Human Morphology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 7, pp. 78–81, July, 1978.     

Cytogenetic analysis of spontaneously activated noninseminated oocytes and parthenogenetically activated failed fertilized human oocytes--implications for the use of primate parthenotes for stem cell production

Purpose: Spontaneous parthenogenetically activated noninseminated oocytes and failed fertilized oocytes after ART activated by puromycin were studied to assess cleavage ability and the cytogenetic constitution of the resulting embryos. Methods: Failed fertilized oocytes were exposed to puromycin, and whenever activation occurred, they were further cultured until arrest of development. FISH was used to assess the ploidy of spontaneous (group A) and induced parthenotes (group B). Results: The mean number of oocytes exposed to puromycin and the percentage and type of activation were identical in IVF and ICSI patients. The more frequent types of activation were one or two pronuclei and one polar body suggesting that retention of the second polar body is a common event after parthenogenetic activation. Conclusions: Retention of the second polar body and chromosome malsegregation were observed after parthenogenetic activation, either spontaneous or induced by puromycin. This means that using parthenogenetic embryos for stem cell research will require great care and attention.     

The effect of puromycin on subepithelial deposits induced by antibodies directed against tubular antigens: a quantitative study.

The mechanism of the GN induced by the intravenous injection of antibodies directed against renal tubular epithelial antigens (RTE) was studied by the use of antibody specifically purified by affinity chromatography. With this reagent RTE antigens could be demonstrated on frozen sections of glomeruli as well as on glomerular cells in culture. The antibodies were radiolabelled, and using paired label techniques glomerular binding could be measured in vivo. In normal rats, approximately 4% of the antibody injected was specifically bound in the glomeruli. Binding was not detectable on proximal tubular brush borders. In rats treated with 100 mg/kg of aminonucleoside of puromycin, a substance known to induce alterations of glomerular epithelial cells, it could be shown that glomerular binding was selectively decreased. These observations indicate that immune deposits in passive Heyman nephitis are formed mainly in situ and that local alteration of the glomerulus can markedly influence deposit formation.