Effects of glycyrrhetinic acid on aminonucleoside nephrosis in rats

The effects of glycyrrhetinic acid (GA), an aglycon of glycyrrhizin extracted from the roots of Glycyrrhizae radix, on puromycin aminonucleoside (PA) nephrosis were studied in rats. Urine protein excretion in female rats (130g–150g) receiving PA (50 mg/kg) alone was significantly elevated on the 2nd day after injection of PA and reached a peak on the 14th day. Urinary protein on the 14th day was reduced to 74% in animals treated with GA (20 mg/kg) starting on the 2nd day after injection of PA. The increase in serum cholesterol and the decrease in serum protein were also suppressed by GA. Observation by electron microscopy revealed that the degree of abnormality in glomerular epithelial cells, i.e. loss or fusion of foot processes, was lower in the rats treated with GA after PA injection than in the rat treated with PA alone. Moreover, pretreatment with GA did not suppress urinary protein excretion but when it was given at the same time as PA and after PA a significant decrease in urinary protein excretion was observed. Correspondence to: H. Abe at the above address     

Nehrin在微小病变型肾病大鼠模型中的表达

目的:观察nephrin在大鼠氨基核苷嘌呤霉素肾病模型(PAN)中的表达变化,探讨其在蛋白尿发生中的作用。方法:通过建立大鼠氨基核苷嘌呤霉素肾病模型,应用光镜、电镜观察肾脏病理改变,应用免疫组织化学技术和RT鄄PCR观察nephrin在不同时间点肾病模型大鼠蛋白水平和mRNA水平表达改变情况。结果:①PAN注射后第1天和第3天,大鼠24h尿蛋白的排泄量较对照组无显著差异;第10天达高峰,较对照组有显著差异(P<0.01);第20天,尿蛋白排泄量逐渐下降,较对照组仍有显著差异(P<0.05);②在PAN肾病模型第3和第10天,透射电镜显示足细胞足突融合;③nephrin蛋白和mRNA水平的表达在大鼠PAN肾病模型第1天即出现下降,第3天出现明显下降,第10天下降到最低点,第20天,随着蛋白尿的恢复nephrin的表达也逐渐恢复;④肾小球足细胞nephrin表达的变化与肾病模型大鼠24h尿蛋白定量的变化呈负相关。结论:①nephrin表达的改变与大鼠氨基核苷嘌呤霉素肾病模型蛋白尿的发生密切相关;②nephrin是判断肾小球足细胞损伤的一个早期重要指标。     

肾八味胶囊对肾病综合征模型小鼠血清IL-6的影响

目的:观察肾八味胶囊对肾病综合征(NS)模型小鼠24h尿蛋白和血清白介素6(IL-6)的影响,以探讨该方对NS的免疫治疗作用机制。方法:给小鼠一次性尾静脉注射嘌呤霉素氨基核苷(Puromycin aminonucleoside,PA)复制NS模型,40只小鼠随机分为模型组、肾八味低剂量组、肾八味高剂量组、至灵胶囊组、生理盐水组,采用BCA法检测各组小鼠24h尿蛋白,采用酶联免疫吸附法检测小鼠血清IL-6的含量。结果:模型组小鼠24h尿蛋白和血清IL-6均较生理盐水组显著升高,中药灌服后能显著降低NS小鼠24h尿蛋白和血清IL-6的含量。结论:降低血清IL-6的含量可能是肾八味胶囊治疗NS的作用机制之一。     

Podocalyxin在大鼠嘌呤霉素肾病模型中的表达及其意义

目的:研究大鼠嘌呤霉素肾病(PAN)模型中podocalyxin的表达,探讨其在蛋白尿发生中的意义.方法:建立PAN模型,应用光镜、电镜观察肾脏病理改变,免疫荧光法检测podocalyxin在肾脏的表达与分布.结果:①PAN 4 d组大鼠24 h尿蛋白量较对照组升高(P＜o.01),PAN 7 d组达高峰(P＜0.001),于28 d逐渐下降至正常.②透射电镜显示PAN 4 d组大鼠足突部分融合,PAN 7 d组大鼠足突广泛融合,PAN 28 d组大鼠足突形态有所恢复.③免疫荧光显示podocalyxin在肾小球表达,PAN 4 d组podocalyxin的表达较对照组降低(P＜0.05).PAN 7 d组表达明显低于对照组(P＜0.01).PAN 28 d组podocalyxin的表达明显高于PAN 7 d组(P＜0.01),与蛋白尿产生明显相关.结论:大鼠PAN模型中,podocalyxin的表达减少可引起足突融合,并与蛋白尿产生密切相关.     

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).     

PODOCYTE INJURY IS SUPPRESSED BY 1,25-DIHYDROXYVITAMIN D3 VIA MODULATION OF TRANSFORMING GROWTH FACTOR-β1/BONE MORPHOGENETIC PROTEIN-7 SIGNALLING IN PUROMYCIN AMINONUCLEOSIDE NEPHROPATHY RATS

• 1 Accumulating evidence suggests that vitamin D and its analogues are renoprotective. However, the precise mechanisms and the molecular targets by which active vitamin D exerts its beneficial effects remain obscure. The objective of the present study was to evaluate the effect of active vitamin D on rats with puromycin aminonucleoside (PAN) nephropathy, a model that is characterized by predominant podocyte injury.
• 2 The PAN nephropathy rats were created by a single intravenous injection of 100 mg/kg PAN. Changes in renal pathology and podocyte numbers were observed. Real‐time polymerase chain reaction (PCR) was performed to examine mRNA expression of nephrin, transforming growth factor (TGF)‐β1 and bone morphogenetic protein (BMP)‐7. Protein expression of nephrin, TGF‐β1, BMP‐7 and p‐Smad2/3 and p‐Smad1/5/8 was examined by immunofluorescence, immunohistochemistry and western blotting, respectively. Rats were treated with 1,25(OH)₂D₃ by gastric gavage at a dose of 2.5 µg/kg per day, starting 2 days before PAN injection and continuing throughout the experiment.
• 3 A single injection of PAN induced massive proteinuria and elevated serum creatinine on Day 7, both of which were significantly suppressed by 1,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃). Immunofluorescence and real‐time PCR of the podocyte‐associated protein nephrin revealed reduced and discontinuous staining and this change was reversed by 1,25(OH)₂D₃. In PAN nephropathy rats, TGF‐β1 and p‐Smad2/3 expression was upregulated, whereas that of BMP‐7 and p‐Smad1/5/8 was downregulated. Treatment with 1,25(OH)₂D₃ significantly restored BMP‐7/Smad signalling while suppressing TGF‐β1/Smad signalling.
• 4 In conclusion, 1,25(OH)₂D₃ can ameliorate podocyte damage and proteinuria induced by PAN. The beneficial effects of 1,25(OH)₂D₃ on podocytes may be attributable, in part, to direct modulation of TGF‐β1/BMP‐7 signalling.

     

Alteration of Connexin43 expression in a rat model of obesity-related glomerulopathy

It is accepted that alteration of connexin43 (Cx43) expression in glomeruli is a common pathological response in several forms of kidney diseases. To date, however the change of the Cx43 expression in obesity-related glomerulopathy (ORG) has not been reported. In this study, the alteration of Cx43 expression in the glomeruli of rat with ORG was defined. Five-week-old rats were fed with high-fat diet for 18 weeks to establish the ORG model, then the histological change of glomeruli, the foot process effacement of podocyte, the markers for podocyte injury (nephrin,podocin and WT1) and Cx43 expression in glomeruli were examined respectively. The results demonstrated metabolic disorder, hyperinsulinemia, systemic inflammation and microalbuminuria in ORG rats. There was significant hypertrophy, glomerular expansion and inflammatory cell infiltration in the kidney of ORG rats compared to the control group. Significant foot process effacement of the podocyte in the glomeruli, nephrin loss and density reduction were shown in the ORG rats, and Cx43 expression was significant upregulated in glomeruli of ORG rats compared to the control group. The results indicate the correlation of overexpressed Cx43 with the obesity related renal inflammation and suggest that Cx43 might be a potential target in the development of obesity related glomerulopathy.     

Fluvastatin attenuates the down?regulation of β1 integrin expression in PAN?treated podocytes by inhibiting ROS

Objective To investigate the effect of fluvastatin（FLV）on the expression of β1 integrin in puromycin aminonucleoside (PAN)?treated podocytes and its mechanism. Methods Cultured human podocytes were divided into PAN, different concentrations of fluvastatin（1×10-8 to 1×10-5 mol/L）,SOD, H2O2 groups respectively. Expressions of β1 integrin and reactive oxygen species (ROS) in podocytes were detected by Western blotting and DCFHDA (2’7’?Dichlorofluoresecein 3’6’?diacetate) respectively. The viability of podocyte was determined by MTT colorimetry. Results PAN and H2O2 significantly decreased the expression of β1 integrin and increased the synthesis of ROS in podocytes (P<0.05 respectively). Lower concentration fluvastatin or SOD treatment up?regulated β1 integrin and down?regulated ROS of podocytes induced by PAN (P<0.05 respectively). MTT revealed that lower podocyte viability was found in higher concentration fluvastatin, PAN and H2O2 groups. Lower concentration fluvastatin and SOD could protect podocytes against PAN. Conclusion Fluvastatin attenuates the injury of podocyte induced by PAN and increases the expression of β1 integrin, whose mechanism may be associated with the inhibition of the ROS activity.     

Role of Protein Synthesis on Ethanol Regulation of Adenylyl Cyclase Activity in Wild-Type S49 Murine Lymphoma Cells

Adenylyl cyclase activity was determined in membranes from wild-type S49 murine lymphoma cells that had been exposed to ethanol for 4 hr. Mn-, NaF-, and forskolin-stimulated adenylyl cyclase activities of cells—pretreated with cycloheximide, puromycin, or serum deprivation—were significantly decreased by treatment with 50 mM of ethanol. As demonstrated for Mn-stimulated activity, the decrease was dose-dependent on ethanol and was temporal; a normal activity recovered after 16–24 hr treatment, even in the presence of cycloheximide and ethanol. Studies with a cell-free membrane system of S49 cells revealed a similar activity decrease after treatment of the membranes with ethanol. In contrast, cells treated with 50 mM of ethanol in a regular culture condition showed no decrease in adenylyl cyclase activity over 24 hr. These results indicate that ethanol regulation of adenylyl cyclase activity in S49 cells depends on reduced or impaired protein synthesis.     

Reduced 11beta-hydroxysteroid dehydrogenase activity in experimental nephrotic syndrome.

BACKGROUND: The disease state of the nephrotic syndrome is characterized by abnormal renal sodium retention that cannot be completely explained by a secondary hyperaldosteronism for the following reasons. Firstly, in rats an enhanced sodium retention is observed before proteinuria with intravascular volume depletion occurs. Secondly, in patients with the nephrotic syndrome, volume expansion with hypertension has been reported despite suppression of the renin-aldosterone system. Therefore, another mechanism for sodium retention must be postulated for this disease state. We hypothesize that this mechanism is a reduced 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2) activity, a phenomenon known to cause enhanced access of cortisol or corticosterone to the mineralocorticoid receptor. METHODS: We assessed the 11beta-HSD activity by measuring the urinary ratio of tetrahydrocorticosterone (THB) plus 5alpha-tetrahydrocorticosterone (5alpha-THB) to 11-dehydro-tetrahydrocorticosterone (THA) by gas chromatography-mass spectrometry in rats with puromycin aminonucleoside (PAN)-induced proteinuria and with adriamycin nephrosis. Furthermore, the plasma ratios of corticosterone to 11-dehydrocorticosterone were measured. RESULTS: The urinary ratio of (THB+5alpha-THB)/THA increased in all animals following injection of PAN or adriamycin, indicating a reduced activity of 11beta-HSD. The reduced activity of 11beta-HSD was confirmed by an increased plasma ratio of corticosterone to 11-dehydrocorticosterone. The changes in the glucocorticoid metabolite ratios were already present before significant proteinuria appeared. CONCLUSION: PAN- or adriamycin-treated rats develop proteinuria with a reduced activity of 11beta-HSD, a mechanism contributing to the abnormal sodium retention in nephrotic syndrome.