Phenotypic modulation of parietal epithelial cells of Bowman's capsule in culture

The origin of cobblestone-like polygonal cells (the most numerous in renal glomerular cell culture) remains controversial; they could be either dedifferentiated podocytes or parietal epithelial cells (PECs) of Bowman's capsule. Poor cellular outgrowth from glomeruli devoid of Bowman's capsule (decapsulated glomeruli) hinders podocytes being obtained without contamination of PECs in culture. Since podocytes are easily damaged during the isolation of glomeruli by the conventional sieving method, we devised a gentle isolation method without forced sieving, resulting in substantial numbers of arborized cells growing out from decapsulated glomeruli. The cells were distinctly different from cobblestone-like polygonal cells in their irregular and often arborized shape and extended long cytoplasmic processes that often crossed over adjacent cells. The arborized cells from decapsulated glomeruli showed intense staining for a podocyte-specific marker, podocalyxin, but no staining for markers specific to PECs (pan cadherin), mesangial cells (Thy-1) or endothelial cells (von Willebrand factor, RECA-1), indicating their podocyte origin. Polygonal cells growing out from encapsulated glomeruli were negative for podocalyxin and positive for pan cadherin at the peripheral cell-cell contact. Thus, the cell population from decapsulated glomeruli is distinctly different from that from encapsulated glomeruli, supporting the idea that polygonal cells originate from PECs, although immunocytochemical markers specific to podocytes in vivo such as WT1, synaptopodin, HSP27 and P-31 antigen were expressed significantly in the polygonal cells. Occasionally, large irregular-shaped cells appeared at the periphery of the outgrowths from encapsulated glomeruli. They were similar in shape to the arborized cells from decapsulated glomeruli but were identical in antigenic properties to cobblestone-like polygonal cells and thus may be named "pseudo-arborized cells". We conclude that PECs in culture modulate their phenotype to resemble podocytes.     

Mixed Organic Solvents Induce Renal Injury in Rats

To investigate the injury effects of organic solvents on kidney, an animal model of Sprague-Dawley (SD) rats treated with mixed organic solvents via inhalation was generated and characterized. The mixed organic solvents consisted of gasoline, dimethylbenzene and formaldehyde (GDF) in the ratio of 2∶2:1, and were used at 12,000 PPM to treat the rats twice a day, each for 3 hours. Proteinuria appeared in the rats after exposure for 5–6 weeks. The incidences of proteinuria in male and female rats after exposure for 12 weeks were 43.8% (7/16) and 25% (4/16), respectively. Urinary N-Acetyl-β-(D)-Glucosaminidase (NAG) activity was increased significantly after exposure for 4 weeks. Histological examination revealed remarkable injuries in the proximal renal tubules, including tubular epithelial cell detachment, cloud swelling and vacuole formation in the proximal tubular cells, as well as proliferation of parietal epithelium and tubular reflux in glomeruli. Ultrastructural examination found that brush border and cytoplasm of tubular epithelial cell were dropped, that tubular epithelial cells were partially disintegrated, and that the mitochondria of tubular epithelial cells were degenerated and lost. In addition to tubular lesions, glomerular damages were also observed, including segmental foot process fusion and loss of foot process covering on glomerular basement membrane (GBM). Immunofluorescence staining indicated that the expression of nephrin and podocin were both decreased after exposure of GDF. In contrast, increased expression of desmin, a marker of podocyte injury, was found in some areas of a glomerulus. TUNEL staining showed that GDF induced apoptosis in tubular cells and glomerular cells. These studies demonstrate that GDF can induce both severe proximal tubular damage and podocyte injury in rats, and the tubular lesions appear earlier than that of glomeruli.     

Cellular origin of fibronectin in interspecies hybrid kidneys

The cellular origin of fibronectin in the kidney was studied in three experimental models. Immunohistochemical techniques that use cross-reacting or species-specific antibodies against mouse or chicken fibronectin were employed. In the first model studied, initially avascular mouse kidneys cultured on avian chorioallantoic membranes differentiate into epithelial kidney tubules and become vascularized by chorioallantoic vessels. Subsequently, hybrid glomeruli composed of mouse podocytes and avian endothelial-mesangial cells form. In immunohistochemical studies, cross-reacting antibodies to fibronectin stained vascular walls, tubular basement membranes, interstitium, and glomeruli of mouse kidney grafts. The species-specific antibodies reacting only with mouse fibronectin stained interstitial areas and tubular basement membranes, but showed no reaction with hybrid glomeruli and avian vascular walls. In contrast, species-specific antibodies against chicken fibronectin stained both the interstitial areas and the vascular walls as well as the endothelial-mesangial areas of the hybrid glomeruli, but did not stain the mouse-derived epithelial structures of the kidneys. In the second model, embryonic kidneys cultured under avascular conditions in vitro develop glomerular tufts, which are devoid of endothelial cells. These explants showed fluorescence staining for fibronectin only in tubular basement membranes and in interstitium. The avascular, purely epithelial glomerular bodies remained unstained. Finally, in outgrowths of separated embryonic glomeruli, the cross-reacting fibronectin antibodies revealed two populations of cells: one devoid of fibronectin and another expressing fibronectin in strong fibrillar and granular patterns. These results favor the idea that the main endogenous cellular sources for fibronectin in the embryonic kidney are the interstitial and vascular cells. All experiments presented here suggest that fibronectin is not synthesized by glomerular epithelial cells in vivo.     

Age-related nephropathy in the Fischer 344 rat is associated with overexpression of collagens and collagen-binding heat shock protein 47

To explore the possible role of heat shock protein (HSP) 47 in the age-related renal changes in Fischer 344 (F 344) rats, the expression of collagen-binding HSP47 with various proteins implicated in phenotypic modulation (α-smooth muscle actin, desmin, and vimentin) and fibrosis (type I, type III, and type IV collagens) was examined in young and old F 344 rat kidneys. Male F 344 rats often develop spontaneous nephropathy in old age. Kidneys obtained from 24-month-old F 344 rats showed glomerulosclerosis with marked tubulointerstitial damage including interstitial fibrosis, while no significant histological alteration was found in the kidneys of 6-month-old rats. Immunohistochemical analysis showed an increased accumulation of type I, type III, and type IV collagens in areas of glomerulosclerosis and interstitial fibrosis in old rat kidneys. In kidneys of young rats, collagen-binding HSP47 expression was weak in the glomeruli and occasionally seen in the interstitial cells. In contrast, strong immunostaining for HSP47 was noted in the glomeruli, tubular epithelial cells, and interstitial cells in kidneys of old rats. In addition, phenotypic alterations of mesangial cells and interstitial cells (immunopositive for α-smooth muscle actin), glomerular epithelial cells (immunopositive for desmin), and tubular epithelial cells (immunopositive for vimentin) were found in the kidneys of old F 344 rats. Double immunostaining showed that all these phenotypically altered renal cells express HSP47 and that increased expression of HSP47 was always associated with increased expression of collagens in the old rat kidneys. From the above observations, it is concluded that overexpression of HSP47 by phenotypically altered renal cells might play an important role in the excessive assembly of collagens and could thereby contribute to the glomerulosclerosis and interstitial fibrosis found in kidneys of aged F 344 rats.     

Three-dimensional distributions of desmin and vimentin in cultured hamster cardiomyocytes using the immunogold deep-etching replica technique

The distributions of desmin and vimentin intermediate filaments in cultured hamster heart cells were examined by immunofluorescent microscopy and an immunogold deep-etching replica technique in combination with electron microscopy. Fluorescent studies showed the overall staining patterns of the myocytes as well as the fibroblasts. Monoclonal antibodies (Da, D₃) to desmin showed punctate staining for the myocytes, while polyclonal desmin (pD) stained in a filamentous pattern. Fibroblasts stained strongly with monoclonal anti-vimentin (Va), but did not stain with the desmin probes. Deep-etched immunogold studies confirmed at the ultrastructural level that monoclonal anti-desmin antibodies stain individual intermediate filaments in an intermittent pattern. Monoclonal (D₃) antibody stained the intermediate filaments heavily and continuously at the cell peripheries, while it stained intermittently in the cell body, similar to the Da monoclonal. Monoclonal anti-vimentin stained only intermediate filaments in fibroblasts. Our studies show a heterogeneity of staining within the cultured heart cells when various anti-desmin and anti-vimentin antibodies are used.     

A morphologic and immunofluorescent analysis of primary guinea pig glomerular cell types grown in chemically defined media. Evidence for clonal growth and cell differentiation

Primary kidney guinea pig glomerular cells were successfully grown in chemically defined media containing insulin, transferrin, and fibronectin or glycylhistidyllysine and fibronectin. Morphologic analysis of glomerular cells grown in either of these chemically defined media provided identical results with respect to cell growth properties and cell types involved. Electron microscopic studies of glomeruli early after they had been placed in culture showed definite evidence of "dedifferentiation" of some glomerular cells. Most glomerular cells in later cultures were undifferentiated. However, since electron microscopic analyses of glomeruli in confluent cultures demonstrated that the majority of cells in culture grow from the epithelial side of the glomerular basement membrane, we suggest that these cells were some form of epithelial cell. This conclusion was further strengthened by the fact that cells resembling well differentiated glomerular epithelial cells were seen in cultures of glomeruli grown in chemically defined media; these cells have never been observed in glomeruli grown in calf serum. Fluorescent microscopy of cell stained with the mitochondrial stain rhodamine 123 allowed identification of several glomerular cell types according to distribution, number, and morphology of mitochondria. Similarly, indirect immunofluorescent microscopy studies using antibodies to fibronectin or laminin provided evidence that glomerular cells separated into cell types according to mitochondrial staining properties were unique biochemically. Using these histochemical criteria it was possible to demonstrate that certain of the glomerular cell types could be selectively grown by addition of the enzyme galactose oxidase to the media. Analysis of our morphologic and histochemical results suggests the possibility that clonal growth and differentiation of glomerular epithelial cells occurs when glomeruli are placed in chemically defined media, and our results are compatible with the hypothesis that either "stem cells" or "dedifferentiated" cells are the primary cells dividing in culture.     

Lipoxygenase activity in rat kidney glomeruli, glomerular epithelial cells, and cortical tubules

We examined the possibility that renal glomerular and cortical tubular tissue has lipoxygenase activity in addition to the well established cyclo-oxygenase pathway of arachidonic acid metabolism. Homogenized rat kidney glomeruli, in the presence of meclofenamate (33 microM) and divalent cation ionophore A23187 (3 microM), metabolized octatritiated arachidonic acid to 12-hydroxyeicosatetraenoic acid and lesser amounts of 80 and/or 9-hydroxyeicosatetraenoic acid. These products were identified by thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectroscopy. In order to rule out the synthesis of hydroxylated fatty acids by platelets and leukocytes entrapped in the glomeruli, we studied lipoxygenase products in glomerular epithelial cells after 9 days in cell culture. Homogenized glomerular epithelial cells converted octatritiated arachidonic acid to 12-hydroxyeicosatetraenoic acid solely. The lipoxygenase activity in cortical tubules was substantially less than in glomeruli and only 12-hydroxyeicosatetraenoic acid was synthesized. The production of hydroxyeicosatetraenoic acid by lipoxygenase inhibitors, nordihydroguaiaretic acid, 5,-homogenized glomeruli, glomerular epithelial cells, and cortical tubules was inhibited by three 8,11,14-eicosatetraynoic acid, and 1-phenyl-3-pyrazolidone. These data demonstrate that there is lipoxygenase activity in rat kidney glomeruli, glomerular epithelial cells and to a lesser extent cortical tubules, and may imply a role of the lipoxygenase products in the regulation of normal glomerular function and inflammatory disease of the kidney.     

Metalloprotease meprin beta in rat kidney: glomerular localization and differential expression in glomerulonephritis

Meprin (EC 3.4.24.18) is an oligomeric metalloendopeptidase found in microvillar membranes of kidney proximal tubular epithelial cells. Here, we present the first report on the expression of meprin beta in rat glomerular epithelial cells and suggest a potential involvement in experimental glomerular disease. We detected meprin beta in glomeruli of immunostained rat kidney sections on the protein level and by quantitative RT-PCR of laser-capture microdissected glomeruli on the mRNA level. Using immuno-gold staining we identified the membrane of podocyte foot processes as the main site of meprin beta expression. The glomerular meprin beta expression pattern was altered in anti-Thy 1.1 and passive Heymann nephritis (PHN). In addition, the meprin beta staining pattern in the latter was reminiscent of immunostaining with the sheep anti-Fx1A antiserum, commonly used in PHN induction. Using Western blot and immunoprecipitation assays we demonstrated that meprin beta is recognized by Fx1A antiserum and may therefore represent an auto-antigen in PHN. In anti-Thy 1.1 glomerulonephritis we observed a striking redistribution of meprin beta in tubular epithelial cells from the apical to the basolateral side and the cytosol. This might point to an involvement of meprin beta in this form of glomerulonephritis.     

Metalloprotease Meprinβ in Rat Kidney: Glomerular Localization and Differential Expression in Glomerulonephritis

Meprin (EC 3.4.24.18) is an oligomeric metalloendopeptidase found in microvillar membranes of kidney proximal tubular epithelial cells. Here, we present the first report on the expression of meprinβ in rat glomerular epithelial cells and suggest a potential involvement in experimental glomerular disease. We detected meprinβ in glomeruli of immunostained rat kidney sections on the protein level and by quantitative RT-PCR of laser-capture microdissected glomeruli on the mRNA level. Using immuno-gold staining we identified the membrane of podocyte foot processes as the main site of meprinβ expression. The glomerular meprinβ expression pattern was altered in anti-Thy 1.1 and passive Heymann nephritis (PHN). In addition, the meprinβ staining pattern in the latter was reminiscent of immunostaining with the sheep anti-Fx1A antiserum, commonly used in PHN induction. Using Western blot and immunoprecipitation assays we demonstrated that meprinβ is recognized by Fx1A antiserum and may therefore represent an auto-antigen in PHN. In anti-Thy 1.1 glomerulonephritis we observed a striking redistribution of meprinβ in tubular epithelial cells from the apical to the basolateral side and the cytosol. This might point to an involvement of meprinβ in this form of glomerulonephritis.     

Matrix Metalloproteinase-9 Expression Is Enhanced in Renal Parietal Epithelial Cells of Zucker Diabetic Fatty Rats and Is Induced by Albumin in In Vitro Primary Parietal Cell Culture

As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy. Albumin overload may induce MMP-9 expression and secretion by PECs via the activation of p44/42 MAPK pathway.     

Culture and Characterization of Epithelial Cells from Bovine Choroid Plexus

Epithelial cells were isolated from choroid plexus, which plays a major role in cerebrospinal fluid production and regulation. Incubation of bovine choroid plexuses with pronase released cells which attached to plastic dishes with a plating efficiency of 5%. The cells were predominantly polygonal as judged by phase-contrast microscopy. These polygonal cells undergo limited cell division and survive for 1-2 weeks in culture before being overgrown by fibroblasts. The fibroblastic cells could be selectively removed from the cultures but the addition of 100 microgram/ml cis-hydroxyproline to the medium for several days. The specific activities of three membrane-bound enzymes, gamma-glutamyl transpeptidase, alkaline phosphatase, and leucine aminopeptidase were compared in selective cultures of polygonal cells and fibroblasts. Polygonal cells were found to have 4-5 times the gamma-glutamyl transpeptidase of fibroblasts, whereas fibroblasts have 2-3 times the alkaline phosphatase of polygonal cells. Leucine aminopeptidase levels in the two cultures were roughly equivalent. The polygonal cells rapidly lost gamma-glutamyl transpeptidase activity over a 4-day period in culture but acquired increased levels of leucine aminopeptidase. Alkaline phosphatase remained roughly constant. Under similar conditions fibroblasts showed a 3- to 4-fold increase in the specific activities of all three enzymes; these changes coincided with a substantial increase in cell density. Based on morphology, resistance to cis-hydroxyproline, absence of antihemophilic factor antigen, and enzymatic characteristics, we believe the polygonal cells to be of epithelial origin.     

Baby hamster kidney (BHK-21/C13) cells can express striated muscle type proteins

When baby hamster kidney (BHK-21/C13) cell lines are subjected to low-serum medium, cell morphology changes from polygonal to elongated and occasionally fusion of cells is also observed. BHK-21 cells initially growing in Eagle's modified minimum essential medium (EMEM) containing 10% newborn bovine serum were induced to differentiate by changing the culture medium after the cells had grown to confluency. After this point the cells were grown in a low-serum medium (EMEM with 2% normal horse serum), for at least 4 days. The expression of different muscle-specific proteins (desmin, titin and skeletal muscle myosin) and of tropomyosins was studied in both polygonal and elongated BHK-21 cells using the indirect-immunofluorescence assay, two-dimensional (2D)-gel electrophoresis and immunoblotting. Filamentous staining was found with the desmin antisera in the polygonal cells and at all stages of BHK-cell elongation. While no reaction was seen with the titin and myosin antibodies in the polygonal cells, a punctate staining reaction for titin was detected 2 days after medium-change, although the cells had not yet elongated. After 4 days titin was found in a striated pattern. Filamentous staining was seen with the skeletal-muscle-specific myosin antibody at this stage. Confirmatory results were obtained from immunoblotting assays and 2D-gel electrophoresis of cytoskeletal preparations from undifferentiated and differentiated BHK cells. These latter experiments showed the initiation of tropomyosin expression only in the differentiated cells. The positive staining with antibodies to skeletal muscle myosin and titin indicates a striated-muscle nature of the (elongated) BHK-21/C13 cells.     

Total numbers of glomeruli and individual glomerular cell types in the normal rat kidney

Summary Alterations in numbers of glomeruli and glomerular cells occur in various renal disorders. Although values for these parameters have previously been reported for several species, the estimates have often been biased due to assumptions regarding glomerular and/or nuclear size and shape. Other studies have used tedious serial-section reconstruction methods. In the present study, unbiased stereological methods were used to estimate total numbers of glomeruli and individual glomerular cell types in normal rats. The kidneys of seven adult Sprague-Dawley rats were perfused with 4% paraformaldehyde and 1% glutaraldehyde in phosphate buffer and embedded in either glycolmethacrylate (for light microscopy, LM) or Epon/Araldite (for transmission electron microscopy, TEM). Total glomerular number was estimated using an LM physical disector/fractionator combination; the total number of cells per average glomerulus was estimated using an LM optical disector/ Cavalieri combination; and TEM physical disectors were used to count individual cell types. The normal rat kidney was found to contain 31764±3667 (mean±SD) glomeruli. An average glomerulus contained 674±129 cells, of which 181±53 were epithelial cells (podocytes), 248±53 were endothelial cells, and 245±45 were mesangial cells. An average renal corpuscle contained 117±27 parietal epithelial cells. Following sectioning and staining, less than 6.5 h was needed to obtain the above estimates for a single animal, with coefficients of variation (SD as a percent of the mean) ranging from 10% to 25%. The unbiased stereological methods used in the present study constitute an unbiased, precise and cost-efficient set of quantitative tools for assessing glomerular morphology in health and disease.     

The immunoreactivity of glial fibrillary acidic protein in mesangial cells and podocytes of the glomeruli of rat kidney in vivo and in culture

In this study the presence of glial fibrillary acidic protein (GFAP) in kidney is for the first time demonstrated in cryostat sections and cultures of isolated glomerular explants derived from rat kidneys. In double immunolabelling analysis of adult rat kidney sections using antiserum against GFAP and monoclonal antibody (mAb) against vimentin or desmin, the presence of immunoreactivity for GFAP could be observed in the glomerulus of the kidney and vascular cells situated in the peritubular space which expressed vimentin and desmin. Labelling of the sections with absorbed antiserum against GFAP completely abolished the staining in all these cells. The mAb against GFAP, clone GF12.24 which is known to label GFAP both in neural and non-neural cells, recognised its antigen only in the cells located in glomeruli. The investigations performed on early 2- or 3-day-old cultures from glomerular explants revealed different patterns of staining for GFAP in mesangial cells and podocytes: weak filamentous in mesangial cells and a strong non-filamentous perinuclear pattern in podocytes. Due to prominent perinuclear expression in podocytes GFAP may be considered as a marker of these cells. A different pattern of distribution of immunoreactivity for GFAP in podocytes and mesangial cells might be due to function-related posttranslational modifications of GFAP resulting in assembly or disassembly of GFAP filaments. The different pattern of staining for GFAP in the podocytes and mesangial cells, cells which exert a different influence on the capillaries of the glomeruli, suggests a role for GFAP in regulation of the tension and permeability of vascular walls. Previous investigations and present studies hint at GFAP as being a general marker of perivascular cells.     

Kidney glomerular explants in serum-free media. Sequential morphologic and quantitative analysis of cell outgrowths

Guinea pig glomeruli were grown in vitro for 22 days in a serum-free medium composed of Waymouth's MB 752/1 supplemented with sodium pyruvate, nonessential amino acids, antibiotics, insulin, transferrin, selenium, triiodothyronine, and fibronectin (FN), and sequential morphologic and quantitative studies of cell outgrowth were performed. Glomeruli grown in serum-free medium showed preservation of glomerular visceral epithelial cells but extensive necrosis of endocapillary cells (endothelial and mesangial cells). Morphologic analysis demonstrated progressive morphologic changes in cultured glomerular cells; however, most cell types observed in culture appeared to grow from the epithelial side of the glomerular basement membrane. Mitosis was a prominent component of glomerular cell outgrowth in vitro, and total DNA increased slightly during glomerular culture. FN was required for glomerular cell outgrowth, and studies using FN fragments demonstrated that the carboxy-terminal portion of FN was required for whole glomerular attachment. These results are used to develop a model for glomerular cell outgrowth in vitro.     

Glomerular lipidosis in a Syrian hamster of the APA strain

Spontaneous glomerular lipidosis was found in a 12-week-old male Syrian hamster of the APA strain. Lipids in the glomeruli were observed as droplets in a prominently expanded mesangial area and as emboli in a dilated capillary lumen. Lipid deposition was also, but less often, detected in tubular epithelial cells and interstitial cells around the lipid-laden glomeruli. This case of glomerular lipidosis was considered to be closely related to hyperglycaemia and hyperlipidaemia.     

Production and immunohistochemical characterization of monoclonal antibodies directed against renal basement membranes of rats

Basement membranes were separated from rat glomeruli and purified by mild procedures, which led to a highly enriched basement membrane fraction. Here, the production and characterization of five monoclonal antibodies against tubular and glomerular basement membranes are described. These antibodies were analyzed immunohistochemically on frozen sections of rat, bovine, and human kidneys as well as on rat embryos. One monoclonal antibody (BM O II) exclusively recognized the glomerular basement membranes, another one (BM O VII) bound to tubular basement membranes and to Bowman's capsule. Three antibodies (BM O IV, BM M II, BM M III) recognized their antigens in both glomerular and tubular basement membranes as well as in mesangial cells. The BM O II antibody showed a stringent species specificity and bound only to glomerular basement membranes of the rat. The other four antibodies cross-reacted with human and bovine glomerular basement membrane and mesangial antigens; they also bound to other tissues in the developing rat embryo. Antibody binding to specific purified components of the basement membranes such as collagen type IV, laminin, heparan sulphate proteoglycan, and fibronectin was investigated by enzyme-linked immunosorbent assay (ELISA). None of these antibodies reacted with any of these known basement membrane components, indicating that the antibodies may serve as useful tools in future investigations of so far unidentified components of basement membranes.     

Rapid preparation of pure glomeruli and tubular fragments from rabbit kidney : Distribution of alkaline phosphatase

A simple rapid method for separating glomeruli and tubular fragments is described. The alkaline phosphatase activity of the glomeruli based on the tissue proteins, or on the DNA, was about 4%, or 1%, respectively of that of the tubular fraction. Alkaline phosphatase can be used as a chemical marker of the purity of glomerular fractions in studies on the structure, antigenicity, and biochemical function of glomeruli.