LYSOSOMAL IRON ACCUMULATION AND TUBULAR DAMAGE IN RAT PUROMYCIN NEPHROSIS AND AGEING

1. Energy dispersive X-ray spectrometry was used to examine the relationship between proteinuria and increased urinary iron excretion, and structural and functional damage in puromycin nephrosis. 2. After 11–12 days rats treated with puromycin (10 mg/100g, i.v.i.) had greater proteinuria (211.6 ± 35.7 mg/day, mean ± s.e.m.) and urinary iron excretion (15.4 ± 2.2 μg/day) than salinetreated controls (14.5 ± 1.4 mg/day and 1.1 ± 0.2 μg/day, respectively, both P<0.001). 3. On day 13, mean lysosomal iron concentration of proximal tubular cells (306.6 ± 64.5 vs 11.9 ± 8.6 mg%, P<0.001), and proximal tubular cell damage assessed semi-quantitively (1.17 ± 0.10 vs 0.62 ± 0.10, P<0.001) were higher and creatinine clearance (0.15 ± 0.01 vs 0.29 ± 0.02 mL/min perg kidney weight, P<0.001) lower than in control rats. 4. At days 35, 60 and 360 there were no differences in any of the measured parameters between rats treated with puromycin or saline, and in both groups proteinuria, tissue damage and lysosomal iron concentration increased with time. 5. Lysosomal iron accumulation was the only independent predictor of both functional and structural damage. 6. In conclusion, the apparent association between proteinuria and tubulo-interstitial damage in puromycin nephrosis, and with ageing, is best explained by factors associated with accumulation of iron within lysosomes of proximal tubule cells.     

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。     

嘌呤霉素抗性基因捕获载体的构建及在细胞中的功能验证

目的 构建具有嘌呤霉素抗性基因捕获载体,扩大基因捕获载体的应用范围.方法 用经改造的捕获载体(gene trapping vector)稳定转染HepG2.2.15肝癌细胞系,经嘌呤霉素筛选,制作单克隆细胞株.用PCR方法验证该载体的在细胞染色体中的整合,ELISA方法证明捕获载体捕获基因后的细胞的功能改变.结果 嘌呤霉素抗性基因捕获载体整合在HepG2.2.15肝癌细胞的染色体上,并能影响细胞HBsAg和HBeAg的分泌.结论 新构建的嘌呤霉素抗性基因捕获载体能在具有G418抗性的细胞中捕获有意义的目的 基因.     

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.

     

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.     

Adrenomedullin protects against oxidative stress-induced podocyte injury as an endogenous antioxidant.

BACKGROUND: We previously reported that puromycin aminonucleoside (PAN) increased adrenomedullin (AM) secretion and AM mRNA expression in podocytes, through overproduction of oxidative stress. To clarify the cytoprotective role of AM as antioxidative and antiapoptotic substance in podocytes, we investigated the effect of exogenous AM and AM antagonist on PAN-induced apoptosis in conditionally immortalized murine podocytes. METHODS: The expression of AM, RAMP 2 and RAMP 3 was investigated using real-time PCR, western blotting analysis and immunofluorescence microscopy. Reactive oxygen species (ROS) production was measured by CM-H(2)DCFDA fluorescence intensity method. The percentage of apoptotic cells was measured by Hoechst 33342 staining. RESULTS: PAN (100 microg/ml) significantly (P < 0.01) increased ROS production, associated with an increase in apoptosis; the percentage of apoptotic cells is 5.3% + 0.05% (P < 0.01) with 36 h treatment of PAN compared to 0.24 + 0.16% with no treatment. Several antioxidants could markedly reduce PAN-induced apoptosis in cultured podocytes, suggesting that PAN-induced apoptosis might be attributable to the overproduction of ROS. Accordingly, the administration of exogenous AM (10(-6) M) could significantly reduce not only ROS production via a PKA-dependent pathway, but also the resultant apoptosis induced by PAN. AM antagonists, CGRP8-37, augmented PAN-induced apoptosis, associated with increased ROS production, 2.2- and 2.3-Fold, respectively. RAMP 2 and RAMP 3 could be detected in podocytes and glomeruli. CONCLUSIONS: This suggests that ROS-induced up-regulation of AM with PAN could counteract ROS-induced apoptosis, by the suppression of ROS production. Therefore, AM might have the endogenous antioxidant potential to protect against ROS-induced podocyte injury.     

Endogenous asymmetrical dimethylarginine and hypertension associated with puromycin nephrosis in the rat

1. The present experiments were designed to investigate the role of asymmetrical N^G,N^G-dimethyl-L-arginine (ADMA) in causing hypertension associated with the focal and segmental glomerulosclerosis (FSGS) produced by a single bolus of puromycin aminonucleoside (PAN) and successive injection of protamine for 7 days in rats which had undergone unilateral nephrectomy.
2. After the unilateral nephrectomy, and administering PAN and protamine, histological examinations of the kidney revealed a typical FSGS, that is, evident abnormalities including segmental mesangial proliferation, obliteration of glomerular capillary lumens and adhesions between the glomerulus and Bowman''s capsule could be observed. Changes in the glomerular epithelial cells consisted of the swelling with bleb formation.
3. In the FSGS rats, urine volume and urinary protein were significantly (P<0.05 and P<0.005) increased throughout 4-week experimental period, while the creatinine clearance was significantly (P<0.005) and transiently decreased, and recovered 4 weeks later. These changes were associated with the sustained elevation of the systolic blood pressure.
4. ADMA levels in aortic endothelial cells, plasma and urine were significantly (P<0.05 and P<0.005) increased in the FSGS rats, but the level in the kidney remained unchanged.
5. The basal level and net production of cyclic GMP in the aortic vessel wall with endothelium when stimulated by norepinephrine and acetylcholine were significantly (P<0.05 and P<0.01) attenuated in the FSGS rats.
6. There were significant and positive correlations between systolic blood pressure (y) and ADMA levels (x) in endothelial cells (y=4.43x+122.2, r=0.979, P<0.0001), plasma (y=0.10x+71.9, r=0.921, P<0.001) and urine (y=0.48x+126.9, r=0.699, P<0.005), but not significant in the kidney (y=0.06x+102.7, r=0.252, NS).
7. These findings suggest that ADMA as an endogenous inhibitor of NO synthesis may play an important role for the pathogenesis in the hypertension associated with the experimental FSGS in the rat.

     

The inhibitory effects of ginsenoside and quercetin on oxidative damage by puromycin aminonucleoside in rat

This study examined the protective effect of two natural antioxidants, ginsenoside Rb-1 and quercetin, on acute nephrosis induced by puromycin aminonucleoside (PA). The protective action of Rb-1 and quercetin were evidenced by their ability to suppress the formation of phosphatidylcholine hydroperoxide in the plasma, liver and kidney. Another beneficial effect noted from these natural antioxidants was increased glutathione peroxidase activity in the blood. Our results suggest that the severity of PA-induced acute nephrosis can be ameliorated by the antioxidative action of these two flavonoids. © 1998 John Wiley & Sons, Ltd.     

Attenuation of diabetic nephropathy by Chaihuang-Yishen granule through anti-inflammatory mechanism in streptozotocin-induced rat model of diabetics

Ethnopharmacological relevance

Traditional Chinese medical herbs have been used in China for a long time to treat different diseases. Based on traditional Chinese medicine (TCM) principle, Chaihuang-Yishen granule (CHYS) was developed and has been employed clinically to treat chronic kidney disease including diabetic nephropathy (DN). The present study was designed to investigate its mechanism of action in treatment of DN.

Materials and methods

Diabetic rats were established by having a right uninephrectomy plus a single intraperitoneal injection of STZ. Rats were divided into four groups of sham, diabetes, diabetes with CHYS and diabetes with fosinopril. CHYS and fosinopril were given to rats by gavage for 20 weeks. Samples from blood, urine and kidney were collected for biochemical, histological, immunohistochemical and molecular analyses.

Results

Rats treated with CHYS showed reduced 24 h urinary protein excretion, decreased serum TC and TG levels, but CHYS treatment did not affect blood glucose level. Glomerular mesangial expansion and tubulointerstitial fibrosis in diabetic rats were significantly alleviated by CHYS treatment. Moreover, CHYS administration markedly reduced mRNA levels of NF-κB p65 and TGF-β1, as well as decreased protein levels of NF-κB p65, MCP-1, TNF-α and TGF-β1 in the kidney of diabetic rats.

Conclusions

CHYS ameliorates renal injury in diabetic rats through reduction of inflammatory cytokines and their intracellular signaling.     

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。