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1

Electronic Resource

In vitro effects of puromycin aminonucleoside on the ultrastructure of rat glomerular podocytes (1990)

Bertram, John F. ; Messina, Aurora ; Ryan, Graeme B.

Springer

Cell & tissue research 260 (1990), S. 555-563

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ISSN: 1432-0878

Keywords: Glomerulus ; Podocytes ; Puromycin aminonucleoside ; Tissue culture ; Morphometry ; Rat (Sprague-Dawley)

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine

Notes: Summary Puromycin aminonucleoside (PAN)-induced nephrosis in rats provides a model for studying the pathogenesis of severe proteinuric conditions, such as minimal change disease. The present study used scanning (SEM) and transmission (TEM) electron microscopy to investigate the in vitro effects of PAN on rat glomerular podocytes. Slices of rat kidney were incubated for up to 3 days in Medium 199 with Hanks' salts (control) or in medium with PAN. Semiquantitative SEM analysis of glomeruli on the upper surface of kidney slices indicated that incubation with PAN (100 μg/ml and 500 μg/ml) decreased the number of microvilli on podocyte cell bodies (days 1, 2 and 3), increased the number of glomeruli showing flattening of podocyte cell bodies and major processes (days 2 and 3), and increased the number of glomeruli showing surface membrane blebbing on podocyte foot processes (day 3) (p〈0.001 in all cases). TEM morphometry revealed that incubation with 500 μg/ml PAN retarded significantly (p〈0.001 at days 2 and 3) the loss of podocyte foot processes observed in control cultures. Whilst the SEM changes to podocyte ultrastructure largely mimic those seen in PAN nephrosis in vivo, the retardation of foot process loss runs counter to the major TEM change observed in vivo.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00297236

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2

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Total numbers of glomeruli and individual glomerular cell types in the normal rat kidney (1992)

Bertram, John F. ; Soosaipillai, Mary C. ; Ricardo, Sharon D. ; [et al.]

Springer

Cell & tissue research 270 (1992), S. 37-45

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ISSN: 1432-0878

Keywords: Kidney ; Glomerulus ; Stereology ; Morphometry ; Disector ; Quantitative methods, structural ; Rat (Sprague Dawley)

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine

Notes: 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.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00381877

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3

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Light-microscopic immunolocalization of fibroblast growth factor-1 and -2 in adult rat kidney (1996)

Cauchi, Jennifer ; Alcorn, Daine ; Cancilla, Belinda ; [et al.]

Springer

Cell & tissue research 285 (1996), S. 179-187

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ISSN: 1432-0878

Keywords: Key words: Fibroblast growth factor ; Kidney ; Immunohistochemistry ; Glomerulus ; Rat (Sprague-Dawley)

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine

Notes: Abstract. The fibroblast growth factors (FGFs) are a family of conserved polypeptides known to regulate cell differentiation and proliferation. We have used avidin-biotin-enhanced indirect immunohistochemistry to localize FGF-1 and FGF-2 in the rat kidney. The most consistent specific immunostaining pattern is found in paraffin sections from kidneys perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer. Intracellular immunoreactivity for FGF-1 and FGF-2 is co-localized in visceral (podocytes) and parietal (Bowman’s capsule) glomerular epithelial cells, S3 segments of proximal tubules, distal tubules and collecting ducts in the cortex, and thick ascending limbs and collecting ducts in the medulla. Immunoreactivity is also observed within urothelium and the tunica adventitia of large blood vessels. No immunostaining is found in cortical S1 or S2 segments of proximal tubules, in frozen sections prepared from unfixed or 4% paraformaldehyde perfusion-fixed kidneys, or in paraffin sections from Bouin-fixed kidneys. Immersion fixation with 4% paraformaldehyde gives a similar staining pattern in paraffin sections to that achieved with perfusion fixation. However, in paraffin sections fixed with methyl Carnoy’s fixative, immunoreactivity is primarily localized to the tunica media of blood vessels, with little tubular or glomerular immunostaining. Thus, variation in immunolocalization patterns for FGFs can be partially attributed to differences in fixative, preparative technique and antibody specificity.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s004410050635

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4

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Glomerular podocytes in cultured rat kidney slices (1989)

Bertram, John F. ; Messina, Aurora ; Dillane, Peter C. ; [et al.]

Springer

Cell & tissue research 256 (1989), S. 419-429

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ISSN: 1432-0878

Keywords: Kidney ; Glomerulus ; Podocytes ; Tissue culture ; Electron microscopy ; Morphometry ; Rat (Sprague Dawley)

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine

Notes: Summary The ultrastructure of rat glomerular epithelial cells (podocytes) in kidney slices in vitro was examined using qualitative and quantitative electron microscopy. The kidney slices were cultured in Medium 199 with Hanks' salts in a 5% CO2/95% O2 environment for up to 14 days. Few changes in podocyte ultrastructure occurred in the first 12 h of culture, but by 24 h cell bodies were rounded, microvilli were present on all podocyte surfaces, and some foot processes had been replaced by flattened expanses of cytoplasm. These changes were more pronounced by 3 days, when some podocytes had developed pseudopodal extensions and appeared to be migrating from glomeruli onto the slice surface. Podocytes could still be identified after 8, 10 and 14 days of culture, although relatively few glomeruli remained at 14 days. Morphometric methods were used to analyse podocyte shape, volume and surface area during the first 4 days of culture. The most significant change involved loss of foot processes: the number of filtration slits per 100 μm of basement membrane decreased from 211.8 ± 15.0 (mean ± SD) at the commencement of culture, to 55.3 ± 22.6 after 2 days (P 〈 0.001). These data provide baseline information for in vitro studies on the effects of nephrotoxins on podocytes.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00218900

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5

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Counting cells with stereology: Random versus serial sectioning (1990)

Bertram, John F. ; Bolender, Robert P.

New York, NY : Wiley-Blackwell

Journal of Electron Microscopy Technique 14 (1990), S. 32-38

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ISSN: 0741-0581

Keywords: Electron microscopy ; Cell counts ; Nuclear shape ; Section compression ; Lung ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Natural Sciences in General

Notes: Counts of cells and nuclei from sections provide information central to studying structural changes in cells, tissues, and organs. This study considers some of the practical problems associated with counting cells with the newer random and serial sectioning methods of stereology and tests the hypothesis that similar cell counts can be obtained with both random and serial sectioning methods.Using irregularly shaped nuclei from alveolar cells of the goat lung, we compared cell counts derived from random (electron microscopic) and serial sectioning (light microscopic) methods. The results showed that both sectioning methods gave similar cell counts (107/cm3 of parenchyma) for type 1 epithelial cells (5.0 vs. 5.0; P=1.0), type 2 epithelial cells (8.6 vs. 9.8; P=0.42) and interstitial cells (34.6 vs. 33.4; P=0.64), provided that corrections were introduced for sectionrelated biases and that the nuclei of the random sectioning method were corrected for shape. We found counting biases of 5%-7% for nuclear shape and 16% for section compression. These observations support the hypothesis that similar cell counts can be obtained with random and serial sectioning, even when nuclei have irregular shapes.

Additional Material: 3 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jemt.1060140106

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6

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Influence of tissue composition on the final volume of rat liver blocks prepared for electron microscopy (1986)

Bertram, John F. ; Bolender, Robert P. ; Sampson, Paul D.

New York, NY : Wiley-Blackwell

Journal of Electron Microscopy Technique 4 (1986), S. 303-314

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ISSN: 0741-0581

Keywords: Fixation ; Shrinkage ; Swelling ; Stereology ; Morphometry ; Life and Medical Sciences ; Cell & Developmental Biology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Natural Sciences in General

Notes: Methods for measuring and evaluating changes in volume of small tissue blocks prepared for transmission electron microscopy are presented. The results indicate: (1) that some blocks swell and others shrink, and (2) that changes in block volume can be explained by inhomogeneous changes occurring in three different tissue compartments.For stereological studies attempting to extrapolate changes in fixed and embedded tissue to those of fresh tissue, consequences of inhomogeneous volume changes in tissue compartments include a decrease in reliability and an increase in statistical variance of stereological density estimates. Since factors could not be found to correct for the changes in individual tissue compartments, alternative strategies are considered for dealing with the volume artifacts of fixation.

Additional Material: 5 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/jemt.1060040306

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