Iimitations of podocyte adaptation for glomerular injury in puromycin aminonucleoside nephrosis

Glomerular synechiae that occurred in nephrotic rats with a single intraperitoneal injection of puromycin aminonucleoside were analyzed by immunohistochemistry, radiolabeled thymidine (PHI-thymidine) autoradiography, as well as light, electron and immunoelectron microscopy. To discriminate podocytes from parietal epithelial cells (PEC) and monocytes, monoclonal antibodies (mAb) against podocalyxin and ED1 were used. The cell kinetics of glomerular epithelial cells were autoradiographically assessed with isotope labeling procedures before and during nephrosis (co-labeled), and a mAb against proliferating cell nuclear antigen (PCNA). All the cell types except the podocyte of normal kidneys were labelled with rHI-thymidine at different rates. Detachment of degenerated podocytes from the outside of the glomerular basement membrane (GBM) is the first step of synechia, and detached sites are confronted by PEC that were hypertro-phied and frequently radiolabeled. Evidence that podocytes in glomeruli of nephrotic rats can proliferate was shown by the presence of mitoses, ^rHt]-thymidine uptake in the co-labeled experiment, and by PCNA staining, but reepithelialization over bare segments of the GBM with proliferated podocytes is doubtful. It was concluded that glomerular synechia resulted from the limits of podocyte adaptation to glomerular injuries.     

Increased oxidative stress and apoptosis in acute puromycin aminonucleoside nephrosis

Accumulating evidence demonstrates that oxidative stress is one of the underlying mechanisms to induce apoptosis in different biological systems. The aim of this study was to examine the simultaneous presence and correlation between oxidative stress events, apoptosis, apoptosis-associated proteins and monocyte/macrophage infiltration during the course of acute puromycin aminonucleoside nephrosis (PAN). To induce nephrosis, Sprague-Dawley rats were injected intraperitoneally with puromycin aminonucleoside and killed at weeks 1 and 2 of nephrosis. Controls represent animals injected with 0.9% saline solution. Kidney sections were homogenized to measure nitric oxide (NO), malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase activities by appropriate enzymatic and biochemical methods. Renal frozen sections were studied for superoxide anion (O(2) (-)) by a histochemical method, for apoptosis by TUNEL (terminal-deoxynucleotidyl-transferase-mediated dUTP- digoxigenin nick end labelling) and for apoptosis-associated protein expression and monocyte/macrophage infiltration by monoclonal antibodies. Increased renal apoptosis, p53, Bax, Bcl-2 accompanied by increased O(2) (-) and NO generation, lipid peroxidation (MDA) and monocyte/macrophage infiltration were found in nephrotic animals. Renal oxidative stress (O(2) (-), NO and MDA) was correlated with apoptosis, p53 expression, monocyte/macrophage cells and proteinuria. Anti-oxidant molecules (SOD and GSH) remained unchanged apart from a decreased activity of catalase which correlated with glomerular apoptosis. In conclusion, the close correlation between the presence of apoptosis and oxidative events confirms the role of oxidative stress in the apoptosis observed during PAN.     

Cyclosporin reduces proteinuria in rats with aminonucleoside nephrosis.

The effect of cyclosporin (CS) was assessed in Sprague-Dawley rats with puromycin aminonucleoside (PA) nephrosis induced by a single intraperitoneal injection of PA. Three groups of rats were injected intraperitoneally with CS (10 mg/kg body weight) daily, beginning 1 day before PA administration, or 5 or 10 days after PA administration, for 10 days. CS significantly reduced proteinuria in rats with PA nephrosis in comparison with untreated nephrotic controls. After discontinuation of the CS treatment, proteinuria gradually increased, reaching values similar to those in control nephrotic rats. CS pretreatment did not prevent the induction of PA-induced nephrotic syndrome. Light microscopy and assessment of anionic sites in the glomerular basement membrane revealed no differences between normal rats, nephrotic controls, and CS-treated rats. These results show that CS can reduce proteinuria in PA nephrosis, but cannot ameliorate the glomerular changes.     

DMSO potentiates aminonucleoside of puromycin nephrosis in rats

Dimethyl sulphoxide (DMSO), 3 g/kg body weight, administered daily by the intraperitoneal route, potentiated the proteinuria and formation of tubular casts in aminonucleoside of puromycin (PA) induced nephrosis in Sprague-Dawley rats. The effect was evident at 4 as well as 8-9 days following PA administration. In the absence of PA, DMSO did not induce proteinuria or cast formation. The mechanism by which DMSO enhanced proteinuria and cast formation is not known.     

Ultrastructural changes in glomerular epithelial cells in acute puromycin aminonucleoside nephrosis: A study by high-resolution scanning electron microscopy

Ultrastructural changes in the podocytes were studied during the development of, and recovery from, acute puromycin aminonucleoside (PAN) nephrosis using high-resolution scanning electron microscopy (hrSEM) and transmission electron microscopy (TEM). In the process of development of PAN nephrosis, four types of early structural changes were observed before total loss of foot processes: formation of cytoplasmic blebs, masking of filtration clefts, flattening of foot processes, and retraction of foot processes. The masking of filtration clefts visualized by hrSEM corresponded to the multiplication of slit diaphragms and adhesion of foot processes in the TEM findings, and preceded retraction of the foot processes. Changes of podocyte configuration were produced. Recovery from this change of podocyte configuration began as islands of podocyte interdigitation, and was proceeded by expansion of the islands. During recovery, the primary processes were re-established either by retraction or perforation of the thin cytoplasm after the formation of foot processes. We conclude that loss of foot processes begins with the masking of filtration clefts. Recovery from the change in podocyte configuration begins with the formation of new foot processes.     

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.     

Modulation of cytoskeletal organization of podocytes during the course of aminonucleoside nephrosis in rats

By immunoelectron microscopy the modulation of cytoskeletal organization of podocytes during the course of puromycin aminonucleoside-induced nephrosis was examined. In control rats, tubulin and vimentin were present, limited to the podocyte cell body and the major processes. Their distribution in the foot processes was virtually negative. Myosin exhibited the same distribution pattern, albeit much more scattered, with no relation to any podocyte organelles or cell structures. Actin was scattered over the fibrillar zones of the cell body and its processes, including the foot processes. In proteinuric rats, loss of foot processes occurred and the glomerular basement membrane was covered by broad cytoplasmic sheets of podocytes, which contained these four subunits of cytoplasmic filaments. Accompanied by the disappearance of proteinuria, the structural organization of the foot processes was completely restored, in which tubulin, vimentin, and myosin were scarcely observed. Our results confirmed that the loss of foot processes is caused by their retraction, and indicated that the specific localization of the podocytic cytoskeleton contributes to the maintenance of the particular cell shape. Its reorganization may account for the structural modification of podocytes.     

CXCL10 induces the recruitment of monocyte-derived macrophages into kidney,which aggravate puromycin aminonucleoside nephrosis

The mechanism responsible for trafficking of monocyte-derived macrophages into kidney in the puromycin aminonucleoside model of nephrotic syndrome in rats (PAN-NS), and the significance of this infiltration, remain largely unknown. CXCL10, a chemokine secreted in many T helper type 1 (Th1) inflammatory diseases, exhibits important roles in trafficking of monocytes and activated T cells. We hypothesized that induction of circulating interferon (IFN)-γ and glomerular tumour necrosis factor (TNF)-α during PAN-NS would stimulate the release of CXCL10 by podocytes, leading to infiltration of activated immune cells and greater glomerular injury. We found that serum IFN-γ, glomerular Cxcl10 mRNA and intra- and peri-glomerular macrophage infiltration were induced strongly during the late acute phase of PAN-NS in Wistar rats, but not in nude (Foxn1^rnu/rnu) rats lacking functional effector T lymphocytes. Wistar rats also developed significantly greater proteinuria than nude rats, which could be abolished by macrophage depletion. Stimulation of cultured podocytes with both IFN-γ and TNF-α markedly induced the expression of Cxcl10 mRNA and CXCL10 secretion. Together, these data support our hypothesis that increased circulating IFN-γ and glomerular TNF-α induce synergistically the production and secretion of CXCL10 by podocytes, attracting activated macrophages into kidney tissue. The study also suggests that IFN-γ, secreted from Th1 lymphocytes, may prime proinflammatory macrophages that consequently aggravate renal injury.     

CD2AP、F-actin在肾病大鼠中的表达及意义

目的：观察CD2-associated protein（CD2AP）、肌动蛋白微丝（F-actin）在氨基核苷肾病大鼠肾小球中的表达变化及其意义。 方法： 通过建立大鼠氨基核苷肾病模型，采用免疫组织化学、蛋白质印迹技术观察CD2AP在不同时点肾病大鼠肾小球中的表达和分布变化，采用荧光技术检测肾小球F-actin含量的变化。 结果： ①肾小球足细胞CD2AP的表达在肾病模型建立的早期即有下调；在肾病大鼠蛋白尿的高峰期，CD2AP的表达明显下降（P<0.01）；在肾病大鼠的疾病恢复期，CD2AP的表达逐步恢复正常。②肾小球足细胞CD2AP表达量的变化与肾病大鼠24 h尿蛋白的变化呈负相关（r=-0.865，P<0.01）。③在肾病大鼠蛋白尿的高峰期，肾小球F-actin含量明显下降（P<0.05）。④肾小球CD2AP蛋白表达量和肾小球F-actin荧光定量变化呈正相关（r=0.873，P<0.01）。 结论： ①CD2AP、F-actin的表达变化在蛋白尿的发生机制中有着重要作用。②CD2AP可能是判断肾小球足细胞损伤的一个早期重要指标。     

利妥昔单抗在难治性微小病变型肾病综合征中的疗效及分析

目的 回顾性分析利妥昔单抗(rituximab,RTX)治疗难治性微小病变型肾病综合征(minimal-change nephrotic syndrome,MCNS)的疗效和安全性,探讨影响其疗效的可能因素.方法 纳入2018年12月至2020年6月于我院肾内科接受RTX治疗的MCNS患者23例,根据发病年龄分为儿童组...     

An ultrastructural study of rat glomerular epithelial cells in culture

Cultured, rat glomerular podocytes were examined by electron microscopy and immunohistochemistry in order to determine their origin. The outgrowth of polygonal cells was noted first. Large arboroid cells were occasionally observed around the polygonal cells. Immunostaining of these large arboroid cells for the intermediate filament proteins showed an intensely positive reaction for vimentin, whereas cytokeratin was not detected. In the polygonal cells, however, both cytokeratin and vimentin were sometimes detected. Although scanning electron microscopy did not detect any specific irregularities in podocytes on the surface of the glomerulus, developing polygonal cells were observed. Both transmission and scanning electron microscopies revealed the polygonal cells to be similar to Bowman's capsule epithelial cellsin situ. The vascular poles of the cultured glomeruli were always in contact with the culture dish. A residual Bowman's capsule was also observed. These results suggest that polygonal cells originate from the epithelial cells of the residual Bowman's capsule, whereas large arboroid cells arise from podocytes. This study was presented at the 25th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Matsumoto, September 28–30, 1993.     

蟾蜍干预对大鼠肾病模型CD2AP表达的影响

目的 探讨CD2-associated protein（CD2AP）在蟾蜍干预阿霉素肾病模型肾小球中的表达及意义。方法 通过建立阿霉素（ADR）肾病模型，用蟾蜍等干预后，采用免疫组织化学技术观察各组模型大鼠肾小球中CD2AP的表达变化。结果 蟾蜍能使肾小球足细胞CD2AP的表达上调。结论 蟾蜍对肾病大鼠模型病理损伤的修复可能与CD2AP表达上调有关。     

Restoration of the renal tubular epithelium of albino rats in the late stages of sublimate necrotizing nephrosis

Department of Biology, Grodno Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR D. S. Sarkisov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 6, pp. 659–661, June, 1991.     

The significance of caveolin-1 expression in parietal epithelial cells of Bowman's capsule

Aims: To analyse the expression of caveolin‐1 in normal human kidney and during diseases leading to nephrotic syndrome in children and to compare its pattern with those observed in control samples, both human and animal. Methods and results: The study group was composed of 104 children diagnosed with minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), lupus glomerulonephritis (LGN) and Schönlein–Henoch glomerulopathy (SH). The research protocol employed direct immunohistochemical assay with the use of mono‐ and polyclonal antibodies against caveolins. Kidney samples of Wistar rats, wild‐type mice and caveolin‐1‐deficient mice were also analysed. In the control human samples, caveolin‐1 was most abundant in the muscle layer of blood vessels and parietal epithelial cells (PECs). Its expression in PECs was significantly lower in children diagnosed with FSGS and LGN than in those with MCD, SH or in controls. In the control animal tissues, except for knock‐out mice, caveolin‐1 was present in distal convoluted tubules, PECs, endothelial cells and muscle. Conclusions: Caveolae are extremely stable elements of PECs and can be excluded from their cell membrane only in response to the dramatic cell reconstruction observed in FSGS and LGN.     

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.     

Usefulness of enzyme-1 and immunohistochemistry in the diagnosis of juvenile xanthogranuloma

Skin biopsies from two children with juvenile xanthogranuloma were studied. Enzyme- and immunohistochemistry was used to establish the mononuclear phagocytic nature of the proliferating dermal elements. The usefulness of both techniques in the diagnosis and the differential diagnosis of this disease is suggested.     

Adenoid cystic (cylindromatous) carcinoma of the trachea: an ultrastructural study

Adenoid cystic (cylindromatous) carcinoma of the trachea is a rare tumour. The histological features in previously reported cases have been widely variable; only one previous ultrastructural study has been undertaken. Three cases are reported in the present study and the ultrastructural findings are reviewed. The tumours were composed of two types of cells, mature ductal and immature myoepithelial. The cylindromatous stroma appeared as a fibrillar substance which was also found between the undifferentiated myoepithelial cells and appeared to be a product of these cells.