Increased glomerular and urinary malondialdehyde in puromycin aminonucleoside-induced proteinuria in rats

Puromycin aminonucleoside (PAN)-induced proteinuria in rats may be mediated by reactive oxygen metabolites (ROM), which are injurious to several cell components including membrane lipids. Increased malondialdehyde (MDA) production is indicative of lipid peroxidation. We examined if MDA content of glomeruli and its urinary excretion were increased in rats administered PAN. Of three groups of 8 Sprague-Dawley rats each, group 1 served a control, group 2 animals received a single intravenous injection of PAN (5 mg/100 g body weight) and group 3 animals PAN with intraperitoneal injections of dimethylthiourea (DMTU), a free radical scavenger of oxidants such as hydroxyl radicals, for 4 days. The rats were sacrificed on day 8 after PAN injection. Increasing proteinuria, starting on day 4, developed in animals in group 2 but not in the others. The glomerular MDA (nmol/mg protein) in group 2 animals was 2.93±1.91, significantly higher than 0.87±0.63 and 1.26±0.76 in groups 1 and 3, respectively. urinary levels of MDA markedly increased in group 2 rats on day 3 and remained high thereafter, but no such increase occurred in the control animals and those administered PAN with DMTU; the latter was thus protective against PAN toxicity. Our observations support the view that ROM are involved in PAN-induced glomerular injury and that increased urinary MDA excretion can be a marker of ROM-mediated lipid peroxidation.     

Effects of low protein diet in puromycin aminonucleoside-induced nephrotic rats

Wistar rats were injected with puromycin aminonucleoside (PAN) to induce massive proteinuria and were raised on a low protein diet to determine whether the urinary protein excretion might be reduced. Although PAN nephrotic rats fed with a normal protein diet containing 20% protein (group A) did not show any reduction of their urinary protein excretion, PAN rats fed with a low protein diet containing 6% protein (group B) revealed a marked reduction at day 9 of the present study in comparison with group A. The total serum protein levels were low in groups A and B as compared to group C, the non-treated control. However, group A recovered to normal levels at day 13. In terms of the absolute amount of oral protein intake, marked differences were observed between groups B and C, but little difference was observed between groups A and C. The body weight in all three groups was decreased at 8 days after the start of this study. The serum levels of creatinine were elevated to 1.1 +/- 0.3 mg/dl and 1.0 +/- 0.2 mg/dl in groups A and B, respectively, on day 7, but abruptly recovered to normal levels by day 12. These data indicate that the decreased urinary protein excretion in group B might be dependent on the total protein volume intake, and not on the renal function nor serum levels of total protein.     

Evidence suggesting a role for hydroxyl radical in puromycin aminonucleoside-induced proteinuria

A single intravenous injection of puromycin aminonucleoside (PAN) results in marked proteinuria and glomerular morphological changes that are similar to minimal change disease in humans. We examined the effect of hydroxyl radical scavengers and an iron chelator on PAN-induced proteinuria. PAN in a dose of 5 mg/100 g body wt significantly increased urinary protein by day 5 (saline: 15 +/- 2, N = 24: PAN: 63 +/- 17, N = 23, P less than 0.001); the proteinuria rapidly increased thereafter, reaching 216 +/- 34, N = 23 by day 7. Concurrent administration of hydroxyl radical scavengers dimethylthiourea, (DMTU 500 mg/kg followed by 125 mg/kg i.p. twice a day) and sodium benzoate (BENZ, 150 mg/kg followed by 125 mg/kg i.p. twice a day) starting the evening before PAN injection markedly reduced proteinuria throughout the course of the study (urinary protein, mg/24 hours on day 7, mean +/- SEM: PAN: 229 +/- 45, N = 15; PAN + DMTU: 30 +/- 5, N = 18; PAN + BENZ: 80 +/- 18, N = 16. Because of the participation of iron in biological systems to generate hydroxyl radical, we also examined the effect of deferoxamine (DFO, 30 mg/day), an iron chelator, on the PAN-induced proteinuria. Concurrent administration of DFO was also protective. In a second series of experiments, DMTU and DFO (administered as described above and then for two additional days after the PAN) provided marked protection even when they were stopped prior to the onset of proteinuria. The protective effects of two hydroxyl radical scavengers and iron chelator implicate an important role for hydroxyl radical in PAN-induced nephrotic syndrome.     

Blunted renal dopaminergic system activity in puromycin aminonucleoside-induced nephrotic syndrome.

BACKGROUND: A primary tubular sodium handling abnormality has been implicated in the edema formation of nephrotic syndrome. Dopamine synthesized by renal proximal tubules behaves as an endogenous natriuretic hormone by activating D(1)-like receptors as a paracrine/autocrine substance. METHODS: We examined the time courses of the urinary excretion of sodium, protein and dopamine in puromycin aminonucleoside (PAN)-treated and control rats. The rats were sacrificed during greatest sodium retention (day 7) as well as during negative sodium balance (day 14) for the evaluation of renal aromatic l-amino acid decarboxylase (AADC) activity, the enzyme responsible for the synthesis of renal dopamine. Also, the influence of volume expansion (VE) and the effects of the D(1)-like agonist fenoldopam (10 microg/kg bw/min) on natriuresis and on proximal tubular Na(+),K(+)-ATPase activity were examined on day 7. RESULTS: The daily urinary excretion of dopamine was decreased in PAN-treated rats, from day 5 and beyond. This was accompanied by a marked decrease in the renal AADC activity, on days 7 and 14. During VE, the fenoldopam-induced decrease in proximal tubular Na(+),K(+)-ATPase activity was more pronounced in PAN-treated rats than in controls. However, the urinary sodium excretion during fenoldopam infusion was markedly increased in control rats but was not altered in PAN-treated animals. CONCLUSION: PAN nephrosis is associated with a blunted renal dopaminergic system activity which may contribute to enhance the proximal tubular Na(+),K(+)-ATPase activity. However, the lack of renal dopamine appears not to be related with the overall renal sodium retention in a state of proteinuria.     

Effects of FK506 on aminonucleoside-induced nephrotic rats]

We examined the effect of immunosuppressive agent, FK506 (Fujisawa, Co.), on puromycin aminonucleoside (PAN)-induced nephrosis. Single i.p. injection of PAN in a dose of 100 mg/kg was introduced into Munich-Wistar rats weighing about 200 g. Those rats were divided into four groups. PAN-induced nephrosis rats in group 1 (PAN-FK0.1, n = 5), group 2 (PAN-FK0.3, n = 5), group 3 (PAN-FK1.0, n = 5) were treated with i.m. injection of FK506 for 10 days in a dose of 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg, respectively, and rats in group 4 were treated with FK-placebo (PAN-PL, n = 5). The rats in group 5 with Saline+placebo were served as a control (NS-PL, n = 5). Among 5 groups, urinary protein, anionic sites (AS) in GBM, and subsets of peripheral lymphocytes through FACS were compared. After 9 days of PAN injection, the rats in PAN-FK0.1 (160.0 +/- 38.4), PAN-FK0.3 (118.0 +/- 34.4) & PAN-FK1.0 (89.2 +/- 40.0) given FK-506 had significantly less proteinuria in a PAN dose dependent manner, compared to those in NS-PL (349 +/- 86.8 mg/day). The numbers of AS/1000 mmGBM were more attenuated in FK506-treated PAN rats (PAN-FK1.0; 16.2 +/- 3.9) than those in PAN-PL (11.7 +/- 4.4). In related to subset of lymphocytes, increased W3/25 in PAN-PL was regressed in PAN-FK0.1, PAN-FK0.3 & PAN-1.0 after 10 days of PAN-injection. W3/25/OX-8 was significantly higher in PAN-PL (3.6) than those in NS-PL (2.4), but not between PAN-1.0 & NS-PL. These data indicate that the mechanism for therapeutic effect of FK506 on PAN-induced nephrosis includes a revision of abnormal cellular immunity, which attenuates the decrease of AS structure and as a result decrease proteinuria.     

Caffeine augments proteinuria in puromycin-aminonucleoside nephrotic rats

Several studies indicate that increased intrarenal adenosine concentrations may attenuate puromycin-aminonucleoside (PAN)-induced nephropathy in rats. The purpose of this study was to investigate the chronic effects of caffeine, a nonselective adenosine receptor antagonist, on renal function and structure in PAN-induced nephropathy. Animals were randomized to receive drinking water or 0.1% caffeine solution. PAN was administered in two doses to a subset from each group at 1 week (100 mg/kg, s.c.; Purom-1) and 15 wks (80 mg/kg, s.c.; Purom-2) after initiating caffeine treatment (PAN and CAFF-PAN groups). The remaining animals served as time controls (CON and CAFF groups). Renal excretory function was followed for 23 wks. Caffeine consumption significantly augmented PAN-induced proteinuria after both PAN injections (Purom-1 and Purom-2, p<0.05 and p<0.001 respectively; CAFF-PAN vs. PAN). In addition, caffeine potentiated the transient reduction in creatinine clearance (CrCl) induced by PAN. Caffeine consumption for 23 wks significantly reduced CrCl in conscious nephrotic animals (4.76 +/- 0.98 vs. 8.51 +/- 1.55 L/kg/day, CAFF-PAN vs. PAN). Seven days after both PAN injections, increased plasma renin activity was detected in animals that were consuming caffeine as compared with corresponding control groups (CAFF and CAFF + PAN vs CON and PAN, respectively). Eight weeks after the second injection of PAN, acute measures of renal hemodynamic and excretory function were compared in anesthetized animals and renal samples were analyzed for histological changes. In PAN-rats, caffeine treatment for 23 weeks significantly reduced inulin clearance (0.28 +/- 0.09 vs. 0.57 +/- 0.12 mL/min/gr kidney. CAFF-PAN vs PAN, p<0.05), tended to increase renal vascular resistance (59.0 +/- 9.5 vs. 42.9 +/- 5.5 mmHg/mL/min/gr kidney, CAFF-PAN vs. PAN, p < 0.06), potentiated the development of more severe tubulointerstitial damage (tubular atrophy, presence of proteinaceous material, tubular dilatation, interstitial inflammation, interstitial fibrosis), and tended to increase glomerulosclerosis. In conclusion, this study indicates that caffeine adversely affects renal function in PAN-nephrotic rats, and that this effect may be due, in part, to increased activity of the renin angiotensin system.     

Graded effects of proteinuria on HDL structure in nephrotic rats

Nephrotic syndrome is characterized by increased triglycerides resulting from decreased clearance of VLDL and chylomicrons. These triglyceride-rich lipoproteins are structurally altered by interaction with HDL derived from animals with proteinuria and not as a consequence of hypoalbuminemia. HDL isolated from rats with massive proteinuria is depleted in apolipoprotein E (apoE). It is unknown at what threshold of urinary albumin loss HDL structure is altered, and it is unknown what effects proteinuria has on apolipoproteins other than apoE. Two models of albuminuria were used in Sprague-Dawley rats: Adriamycin and passive Heymann nephritis (HN). The adriamycin group was divided into minimal albumin excretion (MAE) and intermediate albumin excretion (MAE, 1 to 40; intermediate albumin excretion, 60 to 210 mg/d per 100 g body wt). Urinary albumin excretion exceeded 300 mg/d per 100 g body wt in the HN rats. HDL apolipoprotein composition was analyzed with SDS-PAGE densitometry and liquid chromatography-time of flight mass spectrometer mass spectrometry. HDL apoA-IV content relative to apoA-I was reduced at all levels of albuminuria (P < 0.0001). ApoE was not reduced in MAE but was significantly reduced in IAE (72%; P < 0.001). By contrast, apoA-II and apoC-III were each significantly increased with increasing UAE. ApoA-IV and apoE were decreased to approximately 10% of control in HDL isolated from rats with HN, whereas apoA-II, apoC-II, and apoC-III were each significantly increased relative to apoA-I. HDL is structurally altered by levels of albuminuria that are insufficient to change serum albumin levels and is progressively altered as albuminuria increases.     

Hypoalbuminemia and proteinuria contribute separately to reduced lipoprotein catabolism in the nephrotic syndrome

BACKGROUND: Hypertriglyceridemia is a result of reduced triglyceride (TG)-rich lipoprotein (TRL) catabolism and occurs in rats with nephrotic syndrome (NS) and in Nagase analbuminemic rats (NARs). While the heparin-releasable lipoprotein lipase (LpL) pool in NAR and in NS is similar, TG levels are significantly greater in NS, suggesting that factors other than reduced LpL alone act in NS but not in NARs. Furthermore, clearance of chylomicrons (CM) and very low-density lipoprotein (VLDL) is normal in vivo in NAR despite low LpL levels. We tested the hypotheses that impaired binding of VLDL and impaired VLDL-high density lipoprotein (HDL) interactions contribute to hyperlipidemia in NS. METHODS: TG and apoB secretion was measured using Triton WR 1339. Clearance of CMs by perfused hearts from NS and NAR was determined. Binding of VLDL from control, NS and NAR to rat aortic endothelial cells (RAECs) was measured prior to and following incubation with HDL from NS, NARs, and control. ApoE, protein, and TG content was determined. RESULTS: TG levels were greatest in NS (516 +/- 95 mg/dL), intermediate in NAR (193 +/- 20), and least in control (97 +/- 16, P = 0.05), while in contrast, TG secretion was least in NS (178 +/- 33 mg/dL/hour) versus 212 +/- 17 in NAR and 294 +/- 15 in control (P < 0.001 vs. NS). Clearance of CMs by NS and NAR hearts was the same and significantly reduced versus control (P < 0.005). Binding of NS-VLDL to endothelial cells was reduced, while NAR-VLDL binding was increased versus control (P < 0.001). Incubation of NS-VLDL with control or NAR HDL increased VLDL binding compared with binding following incubation with NS HDL (P < 0.001). CONCLUSION: Increased TG levels in both NS and NAR are the result of decreased TRL clearance. TG levels are greater in NS because of the presence of a combined defect: (1) a decrease in endothelial-bound LpL that occurs as a consequence of reduced serum albumin concentration, and (2) a defect in VLDL binding to endothelial-bound LpL. This latter defect occurs only in the presence of proteinuria and is conferred by HDL.     

Beneficial effects of orosomucoid on the glomerular barrier in puromycin aminonucleoside-induced nephrosis.

BACKGROUND: In a hitherto unconfirmed report, orosomucoid was reported to ameliorate the nephrotic syndrome induced by puromycin aminonucleoside (PAN) in rats. METHODS: We wanted to test this hypothesis and extend the analysis of the effects on the glomerular barrier. Glomerular filtration rate (GFR), and fractional clearance for albumin (theta(albumin)) and for neutral Ficolls were estimated in cooled isolated perfused kidneys. Modern transport equations were used to estimate glomerular size selectivity and charge selectivity. Also, podocyte morphology was studied. Four groups of rats (4 x n = 8) were administered PAN intraperitoneally and treated daily for 5 days with orosomucoid in two different doses (groups A and B), albumin (group C) or saline (group D). Two additional groups of rats (2 x n = 8) were used as controls and these rats received either saline (group E) or orosomucoid (group F) but no PAN. RESULTS: Treatment with orosomucoid restored podocyte morphology and renal function from the damaging effects of PAN in a dose-dependent manner. GFR was significantly reduced by PAN (groups C and D) when compared with controls (groups E and F). This effect was partly (group A) or completely (group B) reversed by orosomucoid. The theta(albumin) was 0.002+/-0.001 (mean+/-SEM) in controls (group E) and was unaffected by orosomucoid per se (group F). PAN increased theta(albumin) to 0.020+/-0.001 in group C and to 0.021+/-0.001 in group D, while it was significantly less in group A, 0.014+/-0.001, P<0.05. The heterogeneous charged fibre model analysis revealed that PAN reduced the relative volume of negatively charged fibres, phi, from 7.1+/-0.08% (group E) to 48% of this value in groups C and D (P<0.001); phi was 4.5+/-0.04% in group A, 5.3+/-0.44% in group B, and 6.1+/-0.11% in group F. CONCLUSION: High doses of orosomucoid completely normalized the glomerular barrier in six out of eight animals with puromycin-induced nephrotic syndrome. Thus, orosomucoid has a promising therapeutic potential for certain kidney disorders.     

足细胞分子高表达致阿霉素肾病大鼠发生蛋白尿

目的 动态观察足细胞裂孔隔膜复合体分子nephrin、podocin、CD2AP及α-actinin-4在阿霉素肾病大鼠蛋白尿发生发展中的表达变化，探讨其在蛋白尿发生发展中的分子行为及其机制。方法 尾静脉注射阿霉素建立阿霉素肾病大鼠模型，3、7、14、28 d每组处死6只大鼠留取肾脏标本。应用间接免疫荧光染色、实时定量PCR、Western 印迹分别检测各个时间点nephrin、podocin、CD2AP、α-actinin-4的分布、mRNA和蛋白表达量的变化。结果 (1)14 d肾病组大鼠24 h尿蛋白显著高于对照组(P < 0.01)，增高持续到28 d。(2)透射电镜显示14 d肾病组足突不同程度变宽，28 d足突弥漫性融合。(3)与对照组相比，从第7天开始肾病组nephrin和podocin染色从沿肾小球毛细血管袢线样分布向不连续粗颗粒样的分布模式转变；CD2AP节段染色增强区逐渐扩大，有的区域呈斑片状和连续线状增强；α-actinin-4从沿肾小球毛细血管袢均匀的点线样分布向不均匀粗颗粒的分布转变，而且随时间进展这种转变逐渐加重。(4)与对照组相比，肾病组于7 d时nephrin mRNA表达显著增高(P < 0.01)；podocin mRNA表达14 d时显著增高(P < 0.05)，直至28 d(P < 0.05)；CD2AP mRNA表达28 d时显著增高(P < 0.05)。(5)与对照组相比，肾病组nephrin蛋白表达28 d时显著增高(P < 0.05)；podocin蛋白表达于7 d时显著增高(P < 0.05)，而28 d时又显著降低(P < 0.05)；CD2AP蛋白表达于14 d时显著增高(P < 0.05)，直至28 d (P < 0.05)。(6)α-actinin-4 mRNA与蛋白表达在实验过程中未出现明显变化。结论 nephrin、podocin和CD2AP的表达增加及分布异常是导致阿霉素肾病大鼠蛋白尿发生发展的分子机制，而分子表达的增加是足细胞抵抗损伤的一种代偿反应。     

Mechanisms of proteinuria in nephrotic humans

The glomerular capillary wall imposes a remarkably efficient barrier to the passage of proteins the size of albumin and larger. The development of heavy proteinuria signifies impairment of the function of this barrier. Because endogenous proteins of graded size are heterogeneous with respect to their molecular charge and undergo extensive tubular reabsorption, they are not useful for quantifying the extent of barrier dysfunction. An alternative approach is to determine the fractional clearance of uncharged and non-reabsorbable polymers of graded size. When combined with a hydrodynamic theory of solute transport through a heteroporous membrane, the intrinsic properties of healthy and diseased glomerular capillary walls can be inferred. This approach reveals the nephrotic range proteinuria that attends minimal change nephropathy to be associated with impairment of both the size- and charge-selective properties of glomerular capillary walls.     

Effect of probucol on hyperlipidemia in patients with nephrotic syndrome

We have evaluated the effect of the antihyperlipidemic agent probucol on hyperlipidemia in patients with nephrotic syndrome. Twelve patients with long-standing nephrotic syndrome received 500 mg of probucol daily for 12 weeks. Serum total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol were significantly lowered with probucol treatment. There were no differences in urine protein, serum total protein, serum albumin and renal function before and after probucol treatment. No drug-related side effects were observed during our study. These results indicated that probucol was effective against hyperlipidemia and free from side effects in patients with persistent nephrotic syndrome. The use of probucol is therefore suggested to be advisable when antihyperlipidemic treatment is required in some subgroups of nephrotic syndrome.     

嘌呤霉素肾病大鼠肾小球中nephrin、Podocin和α-actinin与蛋白尿的关系

目的动态观察肾小球足细胞及裂孔隔膜分子nephrin,podocin和α-actinin在嘌呤霉素(puromycinaminonucleoside,PAN)大鼠肾病模型肾组织中表达的时相变化,探讨这些分子间及这些分子与蛋白尿发生的关系。方法用间接免疫荧光染色及实时定量PCR方法,检测PAN注射后12h、1d、36h、2d、5d、10d、15d及20d大鼠肾小球中nephrin,podocin和α-actinin分子分布和表达。结果(1)PAN注射后1d、2d及5d时,尿蛋白量无明显改变;10d时尿蛋白量明显增加(P=0.02);20d时恢复至对照组水平。(2)对照组大鼠肾小球中nephrin和podocin沿肾小球毛细血管袢呈连续线状分布,α-actinin沿肾小球毛细血管袢呈点线状分布。PAN注射1d后,nephrin和podocin的分布即发生改变,表现为断续、非线性分布。nephrin和podocin的分布改变随着尿蛋白的增多而加重,尿蛋白恢复时也逐渐恢复。20d时,α-actinin沿肾小球毛细血管袢呈连续线性分布。(3)免疫荧光定量分析结果表明,在PAN注射后36h(P=0.04)、2d(P=0.03)及5d(P=0.04)时,肾小球中podocin的免疫荧光染色强度明显下降,于第10d降至最低(P=0.006);自15d时逐渐恢复(P=0.007),20d后podocin的免疫荧光强度恢复至对照组水平。nephrin的免疫荧光染色强度在PAN注射第5天后出现下降(P=0.002),持续下降至第10天(P=     

阿霉素肾病大鼠肾组织中血管形成素样蛋白3表达与蛋白尿及高脂血症的关系

目的 动态观察血管形成素样蛋白3(angiopoietin-like protein 3,ANGPTL3)在阿 霉素大鼠肾病模型肾组织中表达水平的时相变化及细胞定位情况,探讨其与微小病变型肾病综 合征蛋白尿及高脂血症之间的关系。方法 给予大鼠一次性尾静脉注射阿霉素建立微小病变型 肾病综合征大鼠模型。采用免疫组织化学染色和斑点-ELISA,检测阿霉素注射后第7天、第14 天、第21天和第28天大鼠肾组织ANGFTL3的表达及分布。分析其与24 h尿蛋白量及血清胆 同醇(Cho)和甘油三酯(TG)的关系。结果(1)阿霉素注射后第14天开始出现大量蛋白尿(P<0.01), 血清白蛋白明显减少(P<0.01),Cho(P<0.01)和TG(P<0.05)明显增高,以上各项指标均在 第28天达高峰。(2)免疫组化定量分析显示阿霉素注射后第14天时,肾小球中ANGPTL3免疫 组化指数增高至同一时间点对照组的1.42倍(P<0.05);第21天时,为同一时间点对照组的 2.48倍(P<0.01),在肾小管中的表达也明显增高,免疫组化指数为同一时间点对照组的2.04 倍(P<0.01);到第28天,其在肾小球和肾小管中的表达均达最高水平,肾小球的免疫组化指 数为同一时间点对照组的3.28倍(P<0.01),肾小管的免疫组化指数为同一时间点对照组的 2.5倍(P<0.01)。(3)ANGPTL3在肾小球中的表达部位主要位于足细胞胞浆,此外,也可见于