Graeme B. Ryan

The effect of epidermal growth factor on wound healing in mice

Abstract The data presented in this paper focus attention on the possible evolutionary advantages of communal licking, based upon the delivery of wound healing factors in the saliva to an immediate local injury. It is suggested that epidermal growth factor (EGF) is one of these factors, as topical application of EGF to a standardized back wound in mice caged separately enhanced wound closure in both control and sialectomized animals. A sex difference in the wound closing response was evident from these studies. The testosterone dependence of EGF synthesis and its action on wound closure as well as its release upon α-adrenergic stimulation, make teleological sense, in a context of an acute response to injury caused by fighting. It is also suggested that prostaglandins released in injured tissue may modulate these acute effects of EGF, as prostaglandin inhibitors prevented EGF-induced closure. Since EGF is known to be a potent mitogen for murine fibroblast and epithelial cell lines, it may also participate in longer term effects integral to wound healing.

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

SummaryAlterations 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.

Uptake of circulating hyaluronic acid by the rat liver

SummaryThe uptake of [3H]acetyl-labelled hyaluronic acid (HA)Abbreviations used in this paper: HA hyaluronic acid; i.v. intravenous was examined in the liver, spleen and kidney of the rat after i.v. injection. 3H-activity was located by light- and electron-microscopic autoradiography after measurement by scintillation counting of tissue digests. In the liver, approximately 90% of the radioactivity was located in the sinusoidal endothelial cells, with autoradiographic grains distributed throughout the cytoplasm; 50% of the grains overlay vacuoles 0.3 to 1.2 μm in diameter. A few grains (4%) were located in Disses space or nearby in the cytoplasm of hepatocytes. No grains were found in Kupffer cells. The remainder were randomly scattered across the sections in a pattern indicating nonspecific background activity. These observations are in accordance with the selective uptake of HA exhibited by dissociated liver cells in vitro. HA concentrations in the spleen and kidney were too low for detection by autoradiography. Splenic concentrations were much lower than in rabbits or mice; in this respect the uptake of circulating HA in the rat resembles that reported for chondroitin 4-sulphate.

Purification and cloning of a corpuscles of Stannius protein from Anguilla australis

The kidneys of teleost fish are associated with tissues containing secretory granules--the corpuscles of Stannius (CS). Electron microscopy indicates that the granules are of a proteinaceous nature and may represent hormones or enzymes of unrecognized physiological and biochemical function. In the present study, two-dimensional gel electrophoresis and electroelution was used to purify the major protein to homogeneity; it is approximately 32,000 Da in the reduced form and glycosylated. From the partial NH2-terminal sequence, a 75-mer oligonucleotide probe was synthesized and used to isolate a cDNA clone from which the complete amino acid sequence of the major CS protein was deduced. Polyclonal antibodies raised against CS homogenates were specific for the CS proteins (confirmed by immunohistochemistry). Hybridization histochemistry was used to confirm the location of the mRNA encoding the isolated protein. Incubation of CS homogenate with eel plasma or ovine renin substrate did not result in any angiotensin-like peptides whereas kidney homogenate did.

Granular juxtaglomerular cells and prorenin synthesis in mice treated with enalapril.

The short-term and long-term effects (for up to 98 days) of the angiotensin converting enzyme inhibitor enalapril were investigated in male and female BALB/c mice. In control animals, separate antisera to renin and its prosequence produced an identical pattern of staining in granular cells of the juxtaglomerular apparatus (JGA) a short distance from the glomerulus. After 1 day of the enalapril treatment there was a decrease in the number of JGA granular cells immunostained with antisera to both renin and its prosequence. Electron microscopy revealed degranulation of mature granules from JGA granular cells. Fusion of granules with the cell membrane was not observed, but numerous membrane-like structures (myelin figures) were identified in the cytoplasm and extracellular space, indicating possible secretion. In addition, the volume proportion of granulated cells in relation to the glomerular volume was decreased, as was renal renin content. With continuing enalapril treatment, separate antisera to renin and its prosequence stained the same granulated JGA cells with equal intensity. The cells so stained increased in number, extending down the wall of the afferent arteriole to cortical radial arteries (interlobular arteries) upstream from the glomerulus. Ultrastructural studies revealed a progressive development of cytoplasmic granulation in JGA granular cells and in smooth muscle cells extending into cortical radial arteries. Furthermore, the volume proportion of granulated cells in relation to the glomerular volume was significantly increased, as was renal renin content. Thus, short-term enalapril treatment in mice provoked rapid secretion of renin via degranulation of mature granules from JGA granular cells. In contrast, long-term enalapril treatment produced a continuing stimulus for renin synthesis, secretion and storage, resulting in an increased thickness of the afferent arteriolar wall. The mechanism for this change appears to be hypertrophy and hypergranulation of granular JGA cells and neogranulation of smooth muscle cells upstream from the glomerulus. Identification of the intrarenal mediators that induce these phenotypic changes presents an interesting challenge.

Distribution of glomerular peripolar cells in different mammalian species

SummaryPeripolar cells are granulated glomerular epithelial cells that form a cuff around the vascular pole of the glomerulus. Quantitation of these cells in 17 species of mammals (including man, several laboratory animals and a variety of other species) indicated that they were detectable by light microscopy in all but one of the mammals that were examined (the Australian hopping mouse). In adult mammals with detectable peripolar cells, the “peripolar cell index” (the percentage of randomly sectioned glomeruli that displayed peripolar cells in histological sections of kidney) ranged from 0.15 (for echidna) to 11.86 (for sheep). Newborn lambs and rats showed strikingly high values (23.30 and 10.76, respectively) compared with their adult counterparts. Using electron microscopy, peripolar cells were observed in all species that were examined, including the Australian hopping mouse. Morphologically, peripolar cells were similar in all species although their size and granule population varied. They showed a predominantly outer cortical glomerular distribution and a close anatomical relationship with the renin-containing myoepithelioid cells. These findings indicate that peripolar cells are present in a wide variety of species and support the view that such cells may play a significant role in the regulation of normal renal function.

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

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.

Renin processing studied by immunogold localization of prorenin and renin in granular juxtaglomerular cells in mice treated with enalapril

SummaryImmunogold techniques were used to investigate renin processing within granular juxtaglomerular cells following short-term (6 h and 1 day) and long-term (4 weeks) enalapril treatment in female BALB/c mice. In control animals, renin protein labelling was localized to all types of granules (proto-, polymorphous, intermediate and mature) and to transport vesicles, whilst prorenin labelling was found in all these sites except mature granules, confirming that active renin is localized to mature granules only. Following short-term enalapril treatment, the exocytosis of renin protein from mature granules was increased. Long-term enalapril treatment resulted in increased numbers of transport vesicles and all types of granules, consistent with increased synthesis and storage of renin. More large intermediate granules contained discrete regions labelled for prorenin. Renin protein was exocytosed from individual and multiple granules, whilst prorenin was exocytosed from protoand intermediate granules. It is concluded that under normal conditions prorenin is secreted constitutively by bulk flow from transport vesicles. On the other hand, active renin is secreted regulatively from mature granules. In conditions of intense stimulation (angiotensin-converting enzyme inhibition treatment), increased synthesis of prorenin leads to enhanced secretion of prorenin by both constitutive and regulative pathways. Under these conditions, the conversion of prorenin to active renin is increased, with increased secretion of active renin occurring in a regulative manner. Furthermore, the localization of prorenin to one discrete region of large intermediate granules leads us to conclude, that cleavage of the prosegment of renin occurs with the transition of intermediate to mature granules.

Qualitative and quantitative analysis of granules in atrial appendage cardiocytes in different physiological states

SummaryAtrial appendage cardiocytes of mammals, including man, contain multiple cytoplasmic granules that vary in number in different physiological states. Using morphologic and comprehensive morphometric techniques, these granules were assessed in Sprague-Dawley rats following dehydration for 5 days, volume-loading by substituting 1% NaCl as drinking water for 7 days, unilateral nephrectomy plus volume-loading for 7 days, and in late term pregnant animals (18–20 days; term ≈21 days). Although principally located in the paranuclear region, granules were observed throughout the sarcoplasm. Cytological features indicative of synthetic activity and granule formation were readily apparent in all groups with the exception of pregnant rats where they were infrequently observed. Granule contents were released by exocytosis and observed in the right appendage of control, dehydrated and nephrectomy/volume-loaded groups and left appendage of volumeloaded animals. Exocytosis was not observed in pregnant animals. By point counting, the proportional volume of cardiocytes occupied by granules (Vv) in controls was significantly greater for right than for left appendage (2.12±0.22% vs 1.29±0.16%; mean±SEM;p<0.05). A significantly similar difference was found for nephrectomy/volume-loaded animals. There was no significant difference inVv for right appendage between the control and experimental groups; for left appendage there was a significant increase inVv to 2.42±0.09% (p<0.05) for volume-loaded animals only. Estimation of the maximum diameter of granule profiles in control animals was 238±9 nm and 230±6 nm for right and left appendages, respectively. The profile diameters in the left appendages of dehydrated (202±9 nm) and pregnant (200±7 nm) animals were significantly (p<0.05) less than those of the control animals. The morphometric findings did not correlate with predictions based upon published biochemical data. In the course of this study, a previously unreported bimembranous, circular to ovoid structure was observed in the cardiocyte sarcoplasm of all animals; the nature and function of this structure is unknown.

ANGIOTENSIN CONVERTING ENZYME INHIBITION IN THE POSTNATAL RAT RESULTS IN DECREASED CELL PROLIFERATION IN THE RENAL OUTER MEDULLA

1. Chronic angiotensin converting enzyme (ACE) inhibition or AT1 antagonism during postnatal development in the rat has been shown to cause renal tubular and vascular damage, particularly in the outer medulla.