Glomerular structural changes in pregnant, diabetic, and pregnant-diabetic rats

Kidneys enlarge both during pregnancy and in diabetes. The enlargement and morphology of glomeruli was studied during pregnancy and in diabetes in order to examine possible similarities, differences, and interactions in the growth in these conditions. Morphometric investigations were performed on glomeruli in pregnant rats, in rats with 2 weeks' diabetes, and in pregnant-diabetic rats. Kidneys were enlarged 22% in the midterm pregnant rats compared with controls, 74% in diabetic rats, and a further 21% in pregnant-diabetic rats. Glomerular volume was enlarged by 26% during midterm pregnancy in normal animals. Diabetes induced an enlargement in glomerular volume of 58% and a further 18% in midterm diabetic animals due to pregnancy. Within the glomerulus, pregnancy in normal animals induced minor non-significant changes. Diabetes induced significant increase in several parameters: mesangial volume and cell volume, capillary and glomerular basement membrane volume, capillary wall surface area, foot process width, filtration slit length, and nuclear number. Pregnancy in diabetic animals induced no significant additional changes. In conclusion, kidney enlargement in pregnancy shows very few glomerular changes in either normal or diabetic animals. Enlargement of glomeruli in diabetes involves hypertrophy and hyperplasia concurrent with several morphological changes within the glomerulus.     

An experimental kidney disease in dogs produced by injection of heterologous antisera to dog tubular fraction 3 antigen.

Glomerular disease characterised by morphological alterations of the glomerular basement membrane and the mesangium was produced in dogs by injections of heterologous anti-dog renal tubular fraction 3 antibody. The renal lesion was characterised by irregular thickening of the glomerular basement membrane, with electron-dense deposits and loose granular material within it.     

Rapid development of renal lesions in diabetic DBA mice infected with the D-variant of encephalomyocarditis virus (EMC-D).

Marked hyperglycaemia and renal lesions developed rapidly in DBA mice infected with 10 plaque-forming units of the D-variant of encephalomyocarditis virus (EMC-D). Renal alterations were demonstrated in the glomeruli, tubular epithelium and small vessels 2 months after infection. Glomerular changes were characterized by mesangial thickening due to an increase of basement membrane-like material in the mesangial matrix. Nodular glomerular lesions were commonly observed 3 months after infection, whereas distinct thickening of the glomerular basement membrane was rarely seen. Besides these glomerular changes, glycogen inclusions in the distal tubular epithelium and medial degeneration in the arterioles were also noticed. The EMC-D-infected DBA mouse appears to be a useful experimental model for the study of human diabetic nephropathy.     

Rapid development of renal lesions in diabetic DBA mice infected with the D-variant of encephalomyocarditis virus (EMC-D)

Marked hyperglycaemia and renal lesions developed rapidly in DBA mice infected with 10 plaque-forming units of the D-variant of encephalomyocarditis virus (EMC-D). Renal alterations were demonstrated in the glomeruli, tubular epithelium and small vessels 2 months after infection. Glomerular changes were characterized by mesangial thickening due to an increase of basement membrane-like material in the mesangial matrix. Nodular glomerular lesions were commonly observed 3 months after infection, whereas distinct thickening of the glomerular basement membrane was rarely seen. Besides these glomerular changes, glycogen inclusions in the distal tubular epithelium and medial degeneration in the arterioles were also noticed. The EMC-D-infected DBA mouse appears to be a useful experimental model for the study of human diabetic nephropathy.     

Nephrotoxicity of combined treatment with cisplatin and gentamicin in the guinea pig: glomerular injury findings

The drugs cisplatin and gentamicin are given consecutively to various cancer patients suffering from infections. Little information exists about the ultrastructural alterations of kidney glomeruli caused by treatment with these drugs. Renal glomeruli of guinea pigs treated with cisplatin alone and in combination with gentamicin were studied by transmission electron microscopy. The findings revealed foci of damage induced by cisplatin and especially by cisplatin/gentamicin in all glomerular components: glomerular capillaries, including their endothelial cells; basement membrane, epithelial podocytes, mesangial cells, and parietal cells of Bowman's capsule. The damage was expressed by endothelial cytoplasmic extrusions into the vascular lumen, thickening and lamination of capillary basement membrane, focal foot process fusion of podocytes, vacuolization in cytoplasm of endothelial cells of epithelial podocytes and of parietal cells, and the presence of lipid bodies and myeloid bodies in all glomerular cell types. Additionally, injurious effects to cytoplasmic organelles such as mitochondria, nuclei, and endoplasmic reticulum were observed. The results indicate that cisplatin alone and in combination with gentamicin is toxic to renal glomerular tissue. Since these drugs were previously found toxic for strial capillaries in the inner ear and since the main glomerular component is the glomerular capillaries, potential vascular damage and vascular complications in different body systems have to be taken into consideration when these drugs are needed in clinical use.     

Nephrotoxicity of Combined Treatment with Cisplatin and Gentamicin in the Guinea Pig: Glomerular Injury Findings

The drugs cisplatin and gentamicin are given consecutively to various cancer patients suffering from infections. Little information exists about the ultrastructural alterations of kidney glomeruli caused by treatment with these drugs. Renal glomeruli of guinea pigs treated with cisplatin alone and in combination with gentamicin were studied by transmission electron microscopy. The findings revealed foci of damage induced by cisplatin and especially by cisplatin/gentamicin in all glomerular components: glomerular capillaries, including their endothelial cells; basement membrane, epithelial podocytes, mesangial cells, and parietal cells of Bowman's capsule. The damage was expressed by endothelial cytoplasmic extrusions into the vascular lumen, thickening and lamination of capillary basement membrane, focal foot process fusion of podocytes, vacuolization in cytoplasm of endothelial cells of epithelial podocytes and of parietal cells, and the presence of lipid bodies and myeloid bodies in all glomerular cell types. Additionally, injurious effects to cytoplasmic organelles such as mitochondria, nuclei, and endoplasmic reticulum were observed. The results indicate that cisplatin alone and in combination with gentamicin is toxic to renal glomerular tissue. Since these drugs were previously found toxic for strial capillaries in the inner ear and since the main glomerular component is the glomerular capillaries, potential vascular damage and vascular complications in different body systems have to be taken into consideration when these drugs are needed in clinical use.     

Glomerular podocytic injury in protein overload proteinuria

Injection of rats with large doses of bovine serum albumin causes proteinuria which may persist long after the period of overload has ended. In order to assess in this model of proteinuria the relative importance of podocytic epithelial changes versus alterations in anionic groups in the glomerular capillary wall a morphological study has been made of animals in which the kidneys were fixed by vascular perfusion or by in situ drip fixation. By transmission electron microscopy, podocytes showed protein droplets, cytoplasmic vacuoles, spreading of epithelial cytoplasm with loss of foot processes, and focal separation of epithelium from the glomerular basement membrane, occasionally with cytoplasmic disruption. Staining with colloidal iron showed no reduction in the density of anionic groups per unit area on epithelial cell surfaces or elsewhere in glomeruli. However, the reduced surface area of epithelial cells caused by the changes to their structure accounts adequately for the less intense glomerular colloidal iron staining evident by light microscopy. Changes in podocyte structure, particularly those leading to focal cytoplasmic defects on the outer surface of the glomerular basement membrane, appear to be more important than loss of glomerular anionic groups for the development of proteinuria in protein overload nephropathy.     

Immunohistochemical localization of extracellular matrix components in human diabetic glomerular lesions.

The immunohistochemical localization of the extracellular matrix was examined in 31 cases with different degrees of human diabetic nephropathy using antisera to human collagen types I, III, IV, V, fibronectin, laminin, and basement-membrane-associated heparan sulfate proteoglycan (HSPG). In normal glomeruli, HSPG was predominantly localized in the glomerular basement membrane and in the mesangium, and to minor extent in the basement membranes of tubules and Bowman's capsule. Collagen IV and laminin were distributed in glomerular basement membrane and mesangium in minor amounts. Interstitial collagens usually do not occur within glomeruli except for collagen V which has a light microscopic glomerular distribution similar to collagen IV. In diabetic diffuse glomerulosclerosis, the enlarged mesangial matrix showed an increased staining reaction for collagen IV, V, laminin, and fibronectin whereas the staining pattern of HSPG was markedly reduced. Early, small nodular lesions in diabetic glomeruli were similarly positive for most of the basement membrane components, whereas HSPG remained absent. With an increase in the diameter of the noduli, however, the staining reaction for all basement membrane components diminished, whereas interstitial collagens V and III, but not collagen I, were present in these noduli in substantial amounts. These initial studies provide evidence that the changes in the glomerular matrix in diabetic nephropathy may be divided into distinct and progressing stages of lesions. The reduced amount of HSPG even in slight, early lesions may represent the morphologic correlate to the impaired filter function of the glomerular basement membrane.     

Ultrastructural study of nephritis induced in Brown Norway rats by mercuric chloride.

Chronic intoxication by mercuric chloride induces a two-phased nephropathy in Brown Norway rats characterized at first by a linear fixation of antiglomerular basement membrane antibody, then by a granular arteriolar and glomerular fixation of IgG superimposed on the linear one. On the 8th and 9th days, concomitant with the glomerular fixation of antiglomerular basement membrane antibodies, electron microscopy studies demonstrated an influx of monocytes into the glomerular and interstitial capillaries and a focal detachment of the glomerular endothelial cells. On the 14th and 15th days, subendothelial heterogeneous material and scattered subepithelial deposits appeared in glomeruli. By the 60th day, no cellular infiltration nor glomerular endothelial alterations persisted. Subepithelial glomerular deposits were seen in half of the animals, and abnormal deposits situated between muscular cells of the arteriolar walls were seen in all of them. These observations suggest that the glomerular fixation of antiglomerular basement membrane antibodies induces transient alterations of the inner aspect of the glomerular capillary wall. Arteriolar and subepithelial glomerular deposits confirm the idea that an immune-complex disease replaces the antiglomerular basement membrane antibody-mediated process. The morphologic expression of the transition between these two mechanisms is evident around the 15th day.     

Effect of cell substrate on antioxidant enzyme activities in cultured renal glomerular epithelium.

The activities of three antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, were monitored in isolated guinea pig glomeruli and primary or subcultured glomerular epithelial cells. Cell injury was assessed by morphologic studies and by measurement of cellular lipid peroxidation (levels of malondialdehyde). Antioxidant enzyme activities were very different in cultured cells than in parent glomeruli. The possible effect of culture substrates (tissue culture plastic, bovine corneal endothelial [BCE] cell basement membrane, and PF-HR-9 endodermal cell basement membrane) on antioxidant enzyme status, cell morphology, and lipid peroxidation was also assessed. Glomerular epithelial cells cultured on the BCE cell basement membrane substrate survived longer and showed less lipid peroxidation than cells cultured on plastic or the HR-9 substrate. Cells cultured on a plastic substrate had substantially less glutathione peroxidase activity than cells cultured on either BCE or HR-9 basement membranes.     

Glomerular structure in lithium-induced chronic renal failure in rats

The effects of long-term lithium administration on glomerular structure and intervention with angiotensin converting enzyme inhibitor (ACEI) were studied in rats. Male Wistar rats were fed a lithium-containing diet (Li) or control diet (C) for 16 weeks postnatally. Li-treated rats developed renal failure, hypertension and proteinuria. During the subsequent 24 weeks, subgroups were treated with ACEI. The kidneys were fixed by perfusion, and tissue blocks were serially cut for estimation of glomerular volume and glomerular characteristics by light microscopy. Mesangial and mesangial matrix volume fractions, surface density of capillary walls, basement membrane thickness and foot process width (FPW) were measured by electron microscopy. Glomerular volume was decreased in Li-rats, with increased intra-individual variation. In all Li-rats, some glomeruli (mean 27%) were abnormal, with severe changes in only three rats. Ultrastructural parameters obtained by systematic sampling of three glomeruli in each rat showed no differences among groups. Among Li-treated animals there was a significant correlation between FPW and albumin excretion per unit filtration surface, and between filtration surface per glomerulus and inulin clearance. In conclusion, long-term lithium administration to newborn rats caused marked changes in glomerular volume which were not associated with measurable changes in structural parameters. No effect of ACEI-treatment was detectable.     

Alterations in proteoglycan metabolism in the nephrotic syndrome induced by the aminonucleoside of puromycin

The synthesis of intact proteoglycans and their glycosaminoglycan (GAG) side chains by isolated rat glomeruli in vitro were studied both at the onset (5 days) and at the point of maximal proteinuria (7 days) of the nephrotic syndrome induced with the aminonucleoside of puromycin. Glomeruli from nephrotic animals incorporated 1.5-fold and 3.0-fold more 35SO4 label into GAG than glomeruli from control animals at 5 and 7 days, respectively, with heparan-35SO4 GAG being responsible for the majority of the increment. Both nephrotic and control incubations contained 60% of the label in the incubation medium, 40% in the glomerular fractions, and less than 1% in the glomerular basement membrane. Glomerular basement membrane from nephrotic rats had no change in their total heparan-35SO4 GAG content. The majority of intact proteoglycan(s) from the glomerular matrix and from the incubation medium of nephrotic and control animals was found in the most buoyantly dense fraction of CsCl gradients (fraction 1). 35S-labeled material isolated from glomeruli of nephrotic animals showed a consistent shift toward lower density gradient fractions, indicating a decrease in their overall carbohydrate to protein ratio. Diethylaminoethyl chromatography of fraction 1 proteoglycan showed a single biphasic peak with the nephrotic rat having an increase in the proportion contributed by the earlier component of the peak. Fraction 1 proteoglycan(s) from the nephrotic experiment was found to have a smaller average hydrodynamic size by Sepharose CL-2B chromatography without a significant change in the corresponding 35S-GAG chain sizes (molecular weight 14,000) by Sepharose CL-6B chromatography. 35S-macromolecules from glomeruli of nephrotic and control rats that appeared in the middle of the CsCl gradients (fraction 3) had similar Sepharose CL-2B elution volumes, whereas the corresponding 35S-GAG chains from incubations of glomeruli from nephrotic animals were smaller. Increased synthesis of heparan sulfate proteoglycan by glomeruli from puromycin aminonucleoside-induced nephrotic rats may be compensatory to loss of another component of the glomerular filtration barrier or may result from abnormal interaction of proteoglycan(s) from nephrotic animals with other glomerular matrix constituents.     

Experimental glomerulonephritis in the mouse. I. The model

Mice immunized with dog glomerular basement membrane in Fruend's complete adjuvant uniformly developed severe renal lesions. A prominent event in many animals appeared to be mononuclear cell accumulation around small renal blood vessels. Glomerular lesions characterized by hypercellularity, thickening of basement membrane, necrosis, and well marked crescent formation were noted. Immunofluorescent and antibody elution studies from the kidneys of animals immunized with dog GBM demonstrated the presence of specific anti-GBM antibody which could be detected in mouse glomeruli 4 weeks after initial immunization.     

Anti-GBM nephritis in the mouse: severe proteinuria in the heterologous phase

Summary Highly reproducible anti glomerular basement membrane (GBM) nephritis has been induced in the mouse after a single injection of rabbit or goat antibody against purified homologous GBM. The severity of albuminuria was closely related to the amount of antibody given. With doses of 4 mg or more, low serum albumin concentrations, sometimes accompanied by ascites and oedema, were observed after 1 week. Glomerular injury was characterized by an initial accumulation of polymorphonuclear granulocytes followed by thrombosis and necrosis, the extent of which defined the outcome of the glomerulonephritis. With high doses of antibody the exudative lesions entered a chronic phase, while at doses lower than 2 mg remission of the lesions occurred. Immunofluorescence studies showed prompt linear fixation of the injected anti-bodies to the glomerular capillary wall, accompanied by immediate binding of C3 in a fine granular pattern. Fibrin deposits appeared at 2 h in some glomeruli, increased thereafter, and were present after one day in more than 90% of the glomeruli in mice that had received 4 mg of antibody. This new reproducible model in the mouse is suited for the study of the relationship between activation of mediator systems, histological lesions, and proteinuria.     

Ultrastructural observations in a case of BK virus nephropathy with viruses in glomerular subepithelial humps

BK virus nephropathy is a known cause of renal transplant dysfunction and failure. The disease is identified by examination of kidney biopsy tissue utilizing histopathological techniques. Ultrastructural examination of two glomeruli revealed pathology within one glomerulus. Glomerular basement membranes contained subepithelial humps of deposit-like material and BK viruses were identified within this material. Viruses were identified within intertubular capillaries. There was evidence of cytoplasmic clearance of viruses from the glomerular basement membrane by podocytes. The findings may be relevant to the investigation of hump formation and antigen clearance in BK virus nephropathy and postinfectious glomerulonephritis.     

Fc receptor-mediated accumulation of macrophages in crescentic glomerulonephritis induced by anti-glomerular basement membrane antibody administration in WKY rats

Anti-glomerular basement membrane (GBM) glomerulonephritis induced in WKY rats is characterized by glomerular accumulation of CD8(+) T cells and monocytes/macrophages, followed by crescent formation. The mechanism of leukocyte accumulation after antibody binding to GBM is still unclear. To unveil an involvement of Fcgamma receptors (FcgammaR) in leukocytes recruitment we examined the expression of FcgammaR in glomeruli and the effects of the administration of F(ab')(2) fragment of anti-GBM antibody or FcgammaR blocking on the initiation and progression of this model. A gradual increase of FcgammaR mRNA expression in glomeruli during the time course of disease suggested their significance in the development of glomerulonephritis. Glomerular lesions and proteinuria were induced only in rats injected with intact IgG of anti-GBM antibody, but not with the F(ab')(2) fragment. In vivo blocking of FcgammaR by administering heat-aggregated IgG led to the decrease of mRNA expression for all types of FcgammaR (types 1, 2 and 3) and a significant amelioration of glomerulonephritis manifestations. By flow cytometry and immunohistochemistry FcgammaR2-expressing cells in glomeruli were identified as macrophages, but not CD8(+) T cells. The expression of FcgammaR1 and 3 was significantly decreased, and that of FcgammaR2 became undetectable in CD8(+) T cell-depleted rats. Thus, CD8(+) T cells may stimulate FcgammaR expression on macrophages, contributing to their glomerular accumulation and injury. These studies provide direct evidence for a crucial involvement of IgG Fc-FcgammaR interaction in glomerular recruitment of macrophages and following induction of anti-GBM glomerulonephritis in WKY rats.     

The participation of macrophages, glomerular procoagulant activity, and factor VIII in glomerular fibrin deposition. Studies on anti-GBM antibody-induced glomerulonephritis in rabbits.

The temporal relationships of macrophage accumulation, glomerular fibrin deposition, the expression of glomerular procoagulant activity (PCA), and Factor VIII antigen deposition were studied in rabbits in which antiglomerular basement membrane antibody-induced glomerulonephritis (anti-GBM GN) developed. The initiation of injury coincided with the accumulation of glomerular macrophages. Glomerular fibrin, assessed by immunofluorescence and by deposition of 125I-fibrinogen, paralleled the development of glomerular crescents and renal impairment. Macrophage ingress clearly preceded the deposition of 125I-fibrinogen within glomeruli. Augmented levels of PCA were present in glomeruli prior to the initiation of fibrin deposition, and peak levels coincided with the peak glomerular macrophage presence. Factor VIII related antigen was apparent late in the disease and was present mainly at the margins of fibrinous crescents. These data demonstrate that accumulation of glomerular macrophages precedes glomerular fibrin deposition in anti-GBM GN. The augmentation of PCA, coincident with the appearance of glomerular macrophages, suggests a role for macrophage PCA in the initiation of fibrin deposition within the glomerular tuft in this model.     

Culture of human glomerular cells.

Human glomeruli were routinely cultured in Waymouth's medium supplemented with insulin and conditioned medium. Three cell types were seen in the culture of both adult and infant kidneys, but the morphology of the glomerular cellular outgrowths depended on the age of the patient from which the kidney was obtained. Cultures of glomeruli from older individuals resulted in more "differentiated" cells, but both adult and infant glomerular cells rapidly became "dedifferentiated" as the length of time in culture increased. Outgrowths of cultured glomeruli did not contain fibroblasts or tubular cells. Finally, synthesis of basement membrane material by these cultured glomerular cells was demonstrated.     

糖尿病小鼠肾小球微血管密度与VEGF表达的研究

目的: 探讨糖尿病小鼠肾小球微血管密度(MVD)变化及其与血管内皮生长因子(VEGF)表达的关系。方法: 链脲佐菌素诱导小鼠糖尿病6周,定期测量对照组与模型组小鼠体重与血糖变化;常规HE染色观察肾脏形态学变化,采用组织细胞病理分析软件测量肾小球直径、周长及面积;应用免疫组织化学方法检测CD34及VEGF在肾小球的表达,计算MVD值及VEGF表达指数。结果: 与对照组相比,模型组小鼠血糖明显升高(P＜0.01),体重显著降低(P＜0.01),肾小球直径、周长及面积明显增大(P＜0.05);肾小球CD34及VEGF表达明显增强(P＜0.01),肾小球MVD显著增高且与VEGF表达呈显著正相关(r=0.9979,P＜0.05)。结论: VEGF可促进糖尿病小鼠肾小球新生血管生成,VEGF表达上调与糖尿病肾脏病变密切相关。     

DAUNOMYCIN-INDUCED NEPHROPATHY IN RATS

A single intravenous injection of daunomycin into rats Induced severe glomerular injury with massive proteinuria. Mesangial thickening due to an increase in the matrix appeared as early as 5 weeks after injection. Focal and segmental glomerular tuft distortion developed by 10 weeks associated with a progressive mesangial change, which was accompanied by detachments of endothelial cells and podocytes from the glomerular basement membrane (GBM) resulting in obliteration of the affected tufts. After 20 weeks, the lesion ultimately progressed to cause diffuse and global glomerular obliteration. Scattered glomeruli also showed frank shrinkage with a mild obliterative change. By observing a number of isolated glomeruli in scanning electron microscopy, it was revealed that podocyte alterations were variable from case to case and foot processes remained discrete in some cases until 10 weeks, despite the presence of marked proteinuria. Anionic sites distributed throughout the GBM and on the surface of podocytes were usually preserved in proteinuric rats as far as evaluated by ruthenium red and colloidal iron stainings. Our results indicate that loss of foot processes and of glomerular anionic sites are not causative factors but consequences of proteinuria.