Impact of cyclosporin on podocyte ZO-1 expression in puromycin aminonucleoside nephrosis rats

Puromycin aminonucleoside (PAN)-induced nephrosis is a well-described model of human idiopathic nephrotic syndrome, but the mechanism of PAN's effect is not completely understood. To investigate whether proteinuria in the PAN model is associated with an alteration of zonula occludens-1 (ZO-1) expression within the glomeruli, and whether cyclosporin A (CsA) has an effect on proteinuria and ZO-1 expression in this model, eighteen Sprague Dawley (SD) rats were assigned into three groups. Twelve rats received a single intraperitoneal injection of PAN (15 mg/100 g). The other six rats received an equal volume of saline (normal control group; control). CsA solution was administered intraperitoneally once a day for 20 days after the PAN injection (n=6, PAN+CsA). The remaining six rats received PAN, but they didn't receive CsA (n=6, PAN). Compared to control rats (35.1+/-5.4 mg/day), the 24-hour urinary protein excretion on day 18 was significantly higher in the PAN rats (1021.9+/-128.9 mg/day, p<0.01), and the CsA treatment partly reversed the increase in proteinuria in the PAN rats (556.4+/-102.3 mg/day, p<0.05). Glomerular ZO-1 protein expressions were significantly increased in the PAN rats as compared to the control group on day 20 (176%, p<0.01). CsA treatment for 20 days in the PAN rats inhibited the increase in ZO-1 protein expression by 71.1% (p<0.05). CsA treatment significantly diminished the glomerular ZO-1 expression in the PAN rats as assessed by immunohistochemistry. CsA treatment significantly reduced proteinuria and the diminished glomerular ZO-1 expression in a PAN nephrosis rat model. These findings suggest the potential role of the slit diaphragm associated proteins in the development of the nephrotic syndrome, and CsA decreased the proteinuria probably by a direct action on the expression of these proteins in podocytes. Further investigations are needed to clarify the role of slit diaphragm associated proteins in the development of PAN nephrosis.     

Alterations in the distribution of plasma fibronectin and the ultrastructure of podocytes in the peripheral glomerular loops in nephrotic rats

 Glomerular distribution of rat plasma fibronectin was examined during the course of puromycin (PAN)- and daunomycin (DM)-induced nephrosis. In control animals, fibronectin was detected in the mesangial matrix and along the glomerular basement membrane (GBM), closely associated with the plasma membrane of glomerular cells. In peripheral loops, immunoprecipitates were preferentially distributed in the laminae rarae externa and interna. Fibronectin was densely precipitated in a glomerulosclerotic lesion induced by DM at 8 weeks after the injection. In peripheral loops, loss and reconstruction of epithelial foot processes occurred in PAN nephrosis but the change was accompanied by negligible perturbation of fibronectin distribution in the lamina rara externa. In contrast, a remarkable decrease of fibronectin was observed in DM nephrosis, unrelated to the presence or absence of foot processes. The decrease in immunoreactivity for fibronectin in the lamina rara externa seemed to have no association with podocyte attachment to or detachment from the GBM. Plasma fibronectin distributed in the lamina rara externa is not directly involved in the modification of podocyte configuration or podocyte attachment, although its spatial distribution may have some functional significance for preserving the ultrastructure of the GBM. Received: 15 April 1998 / Accepted: 22 June 1998     

Immunohistochemical evaluation of podocalyxin expression in glomerulopathies associated with nephrotic syndrome

It is now well established that morphological change of podocytes is closely correlated to the development of proteinuria. The aim of this study was to investigate the role of podocalyxin, a major podocyte protein, in the pathogenesis of glomerulopathies primarily associated with the nephrotic syndrome. Immunohistochemical expression of podocalyxin has been evaluated in 51 renal samples, including healthy controls, patients with podocytopathies (minimal change disease [MCD], focal segmental glomerulosclerosis [FSGS]) and membranous glomerulopathy (MG). A computerized image analysis program has been used. Statistical analysis was performed using analysis of variance and Bonferroni tests. Immunohistochemical expression of podocalyxin has been observed within the podocytes of healthy controls. In MCD, podocalyxin expression was globally reduced despite the normal appearance of the glomeruli. In FSGS, podocalyxin loss was observed in both the segmental sclerotic and the nonsclerotic areas being significantly more prominent in the former. Reduction of podocalyxin in MG was demonstrated for the first time immunohistochemically. The percentage of the stained area was statistical significantly higher in the controls than in each pathologic group. However, among pathologic groups (FSGS, MCD, MG), there was no statistically significant difference. This is one of the few studies investigating podocalyxin immunohistochemical expression in glomerulopathies associated with nephrotic syndrome. The observed reduction in podocalyxin expression suggests that it constitutes a target molecule in nephrotic syndrome pathogenesis regardless of the underlying cause.     

肾小球滤过屏障超微结构改变与Alport综合征蛋白尿的关系

 目的:探讨肾小球滤过屏障超微结构改变与Alport综合征（AS）蛋白尿发生的关系。方法:AS患者35例,男24例,女11例,肾活检的年龄为1.17~24岁。根据尿蛋白结果将患者分为2组：无/间歇性蛋白尿组（13例）和持续性蛋白尿组（22例）。测量AS肾小球基底膜（GBM）变薄、增厚和致密层撕裂分层的长度,计算其各占GBM总长度的百分比;根据平均足突宽度（FPW）= π／4×（∑基底膜长度／∑足突个数）计算患者足突宽度。分析比较2组患者GBM变薄、增厚、致密层撕裂分层比例和平均FPW及其与AS蛋白尿的关系。结果:持续性蛋白尿组GBM增厚、致密层撕裂分层比例和平均FPW较无/间歇蛋白尿组高（均P<0.05）。平均FPW与GBM增厚、致密层撕裂分层及肾活检年龄均呈正相关（均P<0.01）。结论: AS患者GBM病变程度越重,足突融合越严重,蛋白尿越严重,提示AS基底膜异常有可能通过影响足突及裂孔膜的结构和功能导致蛋白尿发生。     

Sequential ultrastructural podocytic lesions and development of proteinuria in serum sickness nephritis in the rat

Summary Serum sickness nephritis was induced in Fisher rats by immunization with egg albumin (EA) and correlations between immune complex deposition, alterations of podocytes and development of proteinuria were analysed. Immunoelectron microscopy showed that EA, rat IgG and C3 were confined to the electron-dense deposits (Ds). From 3 weeks, when significant proteinuria had developed, the subepithelial region was filled with large numbers of Ds on the peripheral capillary wall as well as in the paramesangium. The loss of slit diaphragms and detachment of foot processes overlying Ds were observed and the escape of Ds into urinary space was frequently detected. Morphometric evaluation showed that the volume of subepithelial Ds and the number of the sites of podocytic detachment correlate significantly with the amount of proteinuria. In addition, the native ferritin injected via the abdominal aorta was seen in large amounts in the urinary space near the areas devoid of epithelial covering. The development of podocytic detachment was clearly coincident with the appearance of proteins with a larger molecular weight in urine. From these results, it is suggested that the loss of slit diaphragms and the detachment of podocytes resulting from the progressive accumulation of Ds will allow the leakage of proteins of larger molecular weight across the capillary wall. These podocytic lesions may be one of the main aetiologies for the development of heavy proteinuria in this model.Part of this study was presented at the 78th Annual Meeting of Japan Pathological Society, in Kyoto, March 1989     

An ultrastructural study of the effect of the steroid in puromycin aminonucleoside nephrosis rats

Summary In order to investigate the significance of the histological change in glomerular epithelial cells in minimal change nephrotic syndrome in man (MCNS) and to help in clarifing the mechanism of action of a steroid in this disease, methylprednisolone was administered to rats with puromycin aminonucleoside nephrosis (PAN). This is an experimental nephrosis having a close resemblance morphologically and physiologically, to human MCNS. Morphological changes in the glomerulus were observed ultrastructurally. The administration of the steroid to PAN rats showed remarkable changes including, rapid disappearance of proteinuria in PAN rats in a manner similer to that seen in human MCNS, and significantly faster recovery of changes in glomerular epithelial cells when compared with spontaneous recovery. From the present study, it is clear that the steroid is effective in rapidly restoring the normal shape of glomerular epithelial cells in PAN rats. The filtration barrier in the glomerular capillary wall (GCW) is also thought to have recovered and proteinuria is cured. Based on these considerations, it may be suggested that proteinuria in human MCNS is caused by changes in glomerular epithelial cells, and that the clinical treatment of proteinuria in MCNS is effective when glomerular epithelial cells have functionally recovered.     

Experimental nephrotic syndrome in the rat induced by puromycin aminonucleoside. Plasma and urinary lipoproteins

Nephrotic syndrome (ascites, proteinuria, hypoalbuminemia, and hyperlipidemia) was induced in male Wistar rats by daily administration of puromycin aminonucleoside (20 mg/Kg). Hyperlipidemia was the result of a marked increase of all plasma lipids particularly triacylglycerols (7 to 8-fold the values found in the pair fed control rats). All plasma lipoprotein fractions were increased in nephrotic rats. VLDL increased 14-fold; LDL and HDL were 7- and 6.5-fold respectively above the pairfed control values. The apoprotein pattern of nephrotic VLDL was characterized by a loss of apoC-1 peptide and the presence of two bands in the region of ARP and apoA-1 peptides. SDS polyacrylamide gel electrophoresis indicated an increased content of ARP in nephrotic VLDL. Nephrotic LDL but not control LDL contained peptides entering the running gel in 8 M-urea gels in the region of ARP, A-1 and C peptides. Nephrotic HDL were almost devoided of ARP and apoA-IV peptides whereas control HDL contained significant amounts of these peptides. The urine of nephrotic rats contained a large amount of lipids (21.9 vs 1.5 M × 10^?6/day). Fatty acids, cholesteryl esters, and phospholipids were the major lipids of nephrotic urine. More than 72% of these lipids were isolated by preparative ultracentrifugation into three fractions: Total Low Density Lipoproteins (< 1.050 g/ml), High Density Lipoproteins (1.063 to 1.210 g/ml) and Ultracentrifugal Residue (> 1.210 g/ml) which contained respectively 7.4, 44.4, and 48.2% of the total recovered lipids. Urinary excretion of HDL was much greater than that of TLDL indicating that lipoproteinuria of aminonucleoside induced nephrotic syndrome was selective. The relationship between these findings, previously reported data and the alterations of plasma and urinary lipoproteins in human nephrotic syndrome is discussed.     

CD28/B7-1与儿童肾病综合征

CD28/B7-1是一对参与机体特异性免疫应答极其重要作用的分子.CD28主要存在于T淋巴细胞表面,而B7-1为CD28的配体之一,可表达于肾脏足突细胞.初步研究显示,B7-1分子参与了足突细胞病变的发生发展,而肾脏足突细胞参与了肾病综合征病理免疫反应过程,尤其在微小病变型肾病综合征中起主导作用.国内外学者已经着手相关动物实验及少量临床试验的研究,并取得一定成果.通过阻断CD28/B7-1信号而抑制肾病综合征的发生发展过程,将为儿童肾病综合征的临床治疗提供新的思路.     

Distribution of renal integrin receptors and their ligands in congenital nephrotic syndrome of the finnish type

The aim of this study was to examine the distribution of 1 and v integrins (Ints) and some of their ligands in the kidneys of patients with congenital nephrotic syndrome of the Finnish type (CNF) and in controls using indirect immunofluorescence with monoclonal antibodies. The mesangial reactivity of Int 1 and Int 1 subunits was more variable and an increased glomerular reactivity with Int 3 and Int-6 antibodies was found in CNF kidneys than in controls. Int 2 subunit was either completely missing from or found in significantly lesser amounts in CNF kidney glomeruli. The immunoreactivity for Int v was more variable, fainter and also more granular in CNF samples than in control kidneys. The glomerular reactivity for Int 5 was more diffuse and weaker, and in sclerotic Bowman's capsules more intense in CNF kidneys than in controls. Immunoreactivity for Int 6 was restricted and was comparable in extent in CNF and control kidneys. Of the extracellular matrix components studied, the expression of EDAFn, EDBFn, OncFn, Ln 2 chain, Ln 1 chain and tenascin was increased. This is also seen in several glomerular diseases with inflammation and sclerosis. Immunoreactivity for vitronectin was decreased. Several differences were found in the intensity or location of the immunostaining for the 1 and v Ints and their ligands in CNF kidneys compared with controls, which have not been found in any other proteinuric disease. Disturbed Int expression pattern in CNF may specifically reflect the disturbance of glomerular function caused by the primary defect in this disease.     

Influence of glomerular filtration rate on renal PAH secretion rate in the rat kidney

PAH secretion (T_PAH) was studied in rats at spontaneously occurring glomerular filtration rate (GFR). At saturated transport, T_PAH was found to be correlated to GFR. This relationship was also observed at unsaturated transport where T_PAH depends upon the PAH concentration in arterial plasma. However, no significant correlation between T_PAH and renal PAH load or renal plasma flow rate was found when the effects of GFR were removed by partial correlation analysis. A dependency of T_PAH on GFR explains the correlations found between filtration fraction (FF) and renal PAH extraction (E_PAH) or renal tubular PAH extraction fraction (E_PAH-FF_PAH). Thus, even at low PAH concentration in a. plasma, renal PAH extraction may only be assumed to be constant if the filtration fraction is constant.     

A study of glomerular basement membrane anionic sites and glomerular visceral epithelial cell coat in protein overload proteinuria in the rat

The presence and distribution of anionic sites in the glomerular basement membrane and visceral epithelial cell coat has been demonstrated. No definite decrease in intensity or periodicity of staining of basement membrane particulate sites was seen in protein overload proteinuric animals and only one staining technique employed for electron microscopy (alcian blue 8GX) demonstrated a focal decrease in visceral epithelial cell coat staining in severely damaged glomeruli. A decrease in overall glomerular staining was also demonstrated by quantitative analysis of colloidal iron staining by light microscopy. The findings differ from those described in puromycin aminonucleoside nephropathy and nephrotoxic nephritis. Staining was demonstrated also in other basement membranes, in Bowman's capsule and along interstitial collagen fibres.     

8种肾小球滤过率预测公式对肾细胞癌患者肾功能评价的适用性及相关因素分析

目的 评价8种常用的肾小球滤过率(GFR)预测公式对肾细胞癌患者肾功能评价的适用性,并分析影响预测公式的相关因素。方法 收集蚌埠医学院第一附属医院泌尿外科2017年1月—2018年12月收治的132例肾细胞癌患者的临床资料进行回顾性分析。记录患者术前测得的血清肌酐Scr值、年龄、性别、体质量指数(BMI)、有无合并症、肿瘤T分期等资料。以外源性放射标记物同位素⁹⁹Tc^m-二乙三胺五醋酸 (DTPA) 的肾排泄率所测得的GFR参照值(rGFR)为标准,应用Bland-Altman分析法比较以下8种预测公式计算得出GFR评估值(eGFR)的偏差:改良 MDRD-1、改良MDRD-2,CKD-EPI公式,联合血清肌酐与胱抑素 C的公式,Cockcroft-Gault (C-G) 公式,基于胱抑素 C的公式 1,基于胱抑素C的公式2,简化 MDRD公式。通过单因素分析方法分别观察患者性别、年龄、BMI、T分期及合并症等对不同预测公式的影响。结果 以rGFR为标准比较8种计算公式的95%一致性分析,胱抑素C相关的2种公式均低估了GFR实际水平,其余6种公式均不同程高估了GFR实际水平。eGFR值偏差较小的3种公式依次是CKD-EPI(7.74 mL/min)、血清肌酐与胱抑素 C(7.87 mL/min)以及改良MDRD-1公式(7.98 mL/min),界外百分比最低的3种公式依次为改良MDRD-2(1.98%)、改良MDRD-1(2.48%)、 C-G公式(2.97%);eGFR值偏差最大的公式为:改良MDRD-2(22.22 mL/min)。通过单因素分析显示8种公式计算的eGFR结果在不同性别、BMI及T分期的肾癌患者间差异均无统计学意义(P值均＞0.05)。除胱抑素C-1和胱抑素C-2两种公式计算结果外,其余6种公式计算的eGFR值在不同年龄段结果不同(随年龄的增高均减少),在合并症组较无合并症组低,差异均有统计学意义(P值均<0.05)。结论 CKD-EPI、联合肌酐胱抑素以及改良MDRD-1对于评价肾癌患者适用性较好,影响eGFR准确度的因素是多样的,患者年龄和是否存在合并症对预测公式的一致性影响较大。