Plasma and urinary endothelin-1 in focal segmental glomerulosclerosis

The kidney is an important site of endothelin-1 (ET-1) production and is particularly susceptible to ET-1 action. Infusion of ET-1 in rats induces both functional and morphological alterations in the kidneys. Increased plasma level of ET-1 has been reported in patients with chronic renal failure. However, there are still no reports on the plasma and urinary ET-1 levels in patients with focal segmental glomerulosclerosis (FSGS). In the present study, we have measured the plasma concentration and urinary excretion rate of ET-1 in 15 patients with nephrotic syndrome due to FSGS, and observed the serial changes of plasma and urinary ET-1 in nephrotic rats with FSGS, induced by repeated injection with puromycin aminonucleoside (PAN). ET-1 was measured with radioimmunoassay. The results showed that plasma ET-1 concentration in FSGS patients was significantly higher than in normal controls (P < 0.05), and that urinary ET-1 excretion rate was also significantly higher in FSGS patients than in normal controls (P < 0.01). In FSGS patients, the plasma and urinary ET-1 was significantly correlated (P < 0.05), and the urinary ET-1 excretion rate was significantly correlated with the amount of proteinuria (P < 0.05) and the glomerular sclerosing score (P < 0.01). In the ten rats with PAN-induced FSGS, serial examination showed a significant increase in plasma ET-1 after 8 weeks of injections, while the urinary ET-1 excretion rate showed a biphasic increase that showed a peak after 4 to 6 weeks. The same changes in plasma and urinary ET-1 levels were not observed in control rats injected with normal saline at the same frequency. Our results suggest that ET-1 may be involved in the pathogenesis of FSGS in both humans and rats.     

Effects of tacrolimus on autophagy protein LC3 in puromycin-damaged mouse podocytes

ObjectiveTo investigate the mechanism through which tacrolimus, often used to treat refractory nephropathy, protects against puromycin-induced podocyte injury.MethodsAn in vitro model of puromycin-induced podocyte injury was established by dividing podocytes into three groups: controls, puromycin only (PAN group), and puromycin plus tacrolimus (FK506 group). Podocyte morphology, number, apoptosis rate and microtubule associated protein 1 light chain 3 alpha (LC3) expression were compared.ResultsPuromycin caused podocyte cell body shrinkage and loose intercellular connections, but podocyte morphology in the FK506 group was similar to controls. The apoptosis rate was lower in the FK506 group versus PAN group. The low level of LC3 mRNA observed in untreated podocytes was decreased by puromycin treatment; however, levels of LC3 mRNA were higher in the FK506 group versus PAN group. Although LC3-I and LC3-II protein levels were decreased by puromycin, levels in the FK506 group were higher than the PAN group. Fewer podocyte autophagosomes were observed in the control and FK506 groups versus the PAN group. Cytoplasmic LC3-related fluorescence intensity was stronger in control and FK506 podocytes versus the PAN group.ConclusionsTacrolimus inhibited puromycin-induced mouse podocyte damage by regulating LC3 expression and enhancing autophagy.     

Rac1及Cdc42在肾上腺髓质肽(AM)缓解嘌呤霉素氨基核苷(PAN)所诱导足细胞损伤中作用机制的初步探讨

目的 观察肾上腺髓质肽（adrenomedullin,AM）对嘌呤霉素氨基核苷（puromycin aminonucleoside,PAN）所诱导受损足细胞的作用以及Rac1/Cdc42表达变化,初步探讨其可能机制。方法 将小鼠肾足细胞株分为对照组、PAN处理组（100 μg/mL）、PAN联合AM处理组（10^-7 mol/L）及PAN联合AM和蛋白激酶A（PKA）抑制剂H89（10^-6 mol/L）处理组。经一次性腹腔注射PAN（150 mg/kg）建立大鼠足细胞损伤模型,每天经尾静脉注射AM蛋白（66 μg/kg）进行干预。SDS-PAGE法检测尿蛋白水平,电镜观察足细胞足突宽度变化,双重免疫荧光染色及/或Western blot观察足细胞特异性骨架蛋白（synaptopodin、nephrin）、Rac1及Cdc42的蛋白表达水平,实时定量PCR（qRT-PCR）检测synaptopodin、nephrin的mRNA表达,谷胱甘肽巯基转移酶-拉下实验（GST-pull down assay）法检测Rac1及Cdc42活性变化。结果 PAN在体内、外均显著降低synaptopodin、nephrin的蛋白或/及mRNA表达水平,且显著升高desmin表达;大鼠PAN肾病模型的尿蛋白水平显著升高,足突宽度显著增加;PAN显著降低Rac1、Cdc42总蛋白及活性水平。上述由PAN所介导的效应被AM显著抑制;H89显著阻断AM的体外作用。结论 AM主要通过PKA通路促进受损足细胞的修复,该保护机制与AM调控Rac1/Cdc42表达密切相关。     

甘草甜素及地塞米松干预嘌呤霉素诱导足细胞损伤的体外研究

目的 观察甘草甜素(GL)及地塞米松(DEX)对受损足细胞凋亡的影响,探讨其细胞学作用机制.方法 建立嘌呤霉素(PAN)致足细胞损伤模型,应用不同水平PAN(12.5mg?L-1“、25.0mg?L-1、50.0 mg?L-1、75.0 mg?L-1、100.0 mg?L-1)作用于足细胞,培养48 h后检测细胞凋亡率,选取合适的PAN作用质量浓度.将体外培养小鼠足细胞(MPC5)分为对照组、PAN组、DEX1组、DEX2组、DEX3组、GL1组、GL2组、GL3组,对照组加入等体积RPMI 1640培养液培养;PAN组加入PAN,终质量浓度50 mg?L-1;DEXL、2、3组同时加入PAN(终质量浓度50 mg?L-1)和DEX(终浓度分别为0.1μmol?L-1、1.0 μmol?L-1、10.0 mol?L-1);GL1、2、3组同时加入PAN(终质量浓度50.0 mg?L-1)和GL(终质量浓度分别为400 n1g?L-1、600 mg?L-1、800 mg?L-1),培养48 h.流式细胞仪检测各组细胞凋亡率.结果 50.0 nmg?L-1 PAN诱导足细胞凋亡较对照组明显升高(P＜0.05),75.0 mg?L-1、100.0 nmg?L-1PAN诱导足细胞凋亡率显著升高,大部分足细胞死亡(Pa＜0.01).DEX1组、2组足细胞凋亡率明显低于PAN组(Pa＜0.05),DEX 3组(10.0 μmol?L-1)干预后足细胞凋亡率显著低于PAN组(P＜0.01).不同质量浓度GL组干预后足细胞凋亡率均显著低于PAN组(Pa＜0.01),随GL质量浓度升高,其抗足细胞凋亡作用降低(P＜0.05).结论 PAN可呈剂量依赖性诱导足细胞损伤,50.0 mg?L-1 PAN为诱导合适的质量浓度.GL及DEX对PAN诱导足细胞损伤均有保护作用,DEX呈剂量依赖方式抑制足细胞凋亡,GL发挥抗足细胞凋亡作用与剂量有关.     

Melatonin promotes puromycin-induced apoptosis with activation of caspase-3 and 5'-adenosine monophosphate-activated kinase-alpha in human leukemia HL-60 cells

Melatonin, a naturally occurring molecule, is produced by the pineal gland in a circadian manner to regulate biologic rhythms in humans. Recent studies report that melatonin may be an attractive candidate as an anticancer agent or for combined therapy because of its antioxidant, oncostatic and immunoregulatory activities. In this study, the potentiating effect of melatonin was evaluated on the apoptosis induced by puromycin as an anticancer drug in acute promyelocytic leukemia HL-60 cells. Melatonin did not show significant cytotoxicity against HL-60 cells compared to puromycin. However, melatonin significantly augmented the cytotoxicity of puromycin. Consistently, combined treatment of melatonin and puromycin reduced the expression of anti-apoptotic proteins, such as bcl-2 and bcl-x(L) , and also induced caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage compared to puromycin treatment alone. Furthermore, cell cycle analysis revealed that melatonin promoted puromycin-induced apoptosis by increasing the sub-G1 population, but suppressing G2/M arrest in HL-60 cells. Interestingly, melatonin activated the phosphorylation of 5'-adenosine monophosphate-activated kinase (AMPK) in combination with puromycin. Taken together, our results suggest that melatonin potentiates puromycin-induced apoptosis with caspase-3 and AMPK activation in HL-60 cells, and thus, melatonin treatment can be effectively applied to leukemia treatment as a potential sensitizer for chemotherapeutic agents.     

Multi-glycoside of Tripterygium wilfordii Hook. f. reduces proteinuria through improving podocyte slit diaphragm dysfunction in anti-Thy1.1 glomerulonephritis

Ethnopharmacological relevance

Multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) has been proved clinically effective in reducing proteinuria in chronic kidney disease in China. However, the mechanisms involved are still unclear. In this study we examined the effects of GTW at the different dosages on proteinuria and podocyte slit diaphragm (SD) dysfunction in anti-Thy1.1 glomerulonephritis (GN).

Materials and methods

Rats with anti-Thy1.1 GN were divided into 2 groups, a GTW group and a vehicle group, and sacrificed at 30 min, on day 7, and on day 14 in Experiments 1, 2 and 3, respectively. The administration of GTW at the moderate and high doses was started 3 days before or at the same time of antibody injection till sacrifice. Proteinuria was determined in Experiments 1, 2, and 3. After sacrifice, the staining intensity of SD-associated key functional molecules including nephrin and podocin, podocyte structure, mesangial change, macrophage infiltration, and blood biochemical parameters were examined, respectively. Protein and mRNA expressions of nephrin and podocin in glomeruli were also investigated. Besides, liver histological characteristics were analyzed.

Results

In Experiment 1, GTW pretreatment at the medium dose (75 mg/kg body weight) caused no influence on the induction of anti-Thy1.1 GN and the basal nephrin expression. In Experiment 2, the high dosage (100 mg/kg body weight) of GTW ameliorated proteinuria, the distribution of nephrin and podocin, mesangial proliferation, and the activated macrophage accumulation, as compared with vehicle group (P < 0.05). Additionally, it increased mRNA and protein expressions of nephrin and podocin in glomeruli on day 7, but had no influence on podocyte structure. In Experiment 3, the medium dosage (75 mg/kg body weight) of GTW improved proteinuria, the partial matrix expansion, and the distribution of nephrin and podocin on day 14, as compared with anti-Thy1.1 GN rats (P < 0.05). GTW at the high or moderate dose did not affect hepatic function on day 7 and on day 14.

Conclusions

Podocyte SD dysfunction, such as the disordered distribution and down-regulation of nephrin and podocin expression, is critically involved in the pathogenesis of anti-Thy1.1 GN induced by mAb 1-22-3. The restoration of the distribution and expression of nephrin and podocin by GTW could be an important mechanism by which GTW ameliorates proteinuria and podocyte SD dysfunction.     

雷至胶囊对嘌呤霉素氨基核苷肾病的减毒增效作用

目的:探讨雷至胶囊(雷公藤多苷10 mg.kg-1+女贞子、墨旱莲4 g.kg-1)在治疗嘌呤霉素氨基核苷(PAN)肾病的减毒增效作用。方法:80只雄性大鼠随机分为8组:空白组、模型组、雷至胶囊高、低剂量组、雷公藤多苷高、低剂量组、雷公藤甲素组和缬沙坦组,每组10只。颈静脉注射PAN 100 mg.kg-1建立PAN肾病模型。空白组颈静脉注射等量生理盐水。其余各治疗组造模后第2天开始每日分别ig给药,持续10 d。第11天,处死大鼠,摘取肝肾,用于光镜、免疫荧光及透射电镜检测。结果:一般情况:模型鼠第5天尿量减少、腹水,而浮肿不明显,体重下降。第7～10天腹水明显,24 h尿蛋白增加,死亡率30%(3/10)。病理改变:模型鼠肾小管上皮细胞变性,电镜下,可见足细胞足突融合、消失,而空白组足细胞结构正常。各药物治疗组24 h尿蛋白、血生化、血红蛋白均有不同程度改善,各治疗组血尿素氮轻度升高(P<0.05)。雷至胶囊及雷公藤多苷10 mg.kg-1组降蛋白尿、改善贫血、降低AST疗效优于其余各组,电镜下,雷至胶囊高剂量组足突融合减轻,足突明显恢复。结论:雷至胶囊可能通过降低PAN肾病大鼠蛋白尿、改善贫血、降低AST、减轻足细胞足突融合及肾小管上皮细胞变性等发挥减毒增效作用。