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Biomedical subjects

G B Ryan

Publications and source records attributed to G B Ryan.

At least 19 recordsLinked to original sources
PubMed1997

Embryonic and postnatal development of the rat renal interstitium.

Whilst antihypertensive, structural and functional roles have been proposed for the cells of the renomedullary interstitium in the adult kidney, little is known about its role in renal development. Rat kidneys were studied throughout development, prenatally at gestational ages E14-E21 and postnatally at 0-28 days, by light microscopy, transmission and scanning electron microscopy and following immunocytochemistry. Renomedullary interstitial cells were observed as early as embryonic day E14, forming a loose, orderly network around branches of the ureteric bud. Paralleling the development of the first nephron structures, renomedullary interstitial cells were arranged in a concentric circular manner around collecting ducts. Following tubular and vascular growth from the cortex into the medulla, this arrangement resulted in the characteristic 'rungs of a ladder' appearance of interstitial cells between tubules, blood vessels and the collecting ducts. Renomedullary interstitial cells were closely adherent to basement membranes of tubules, blood vessels and collecting ducts from early in development. Contacts were absent between renomedullary interstitial cells and tubular structures in the process of remodelling, such as the hair-pin bends of the loops of Henle. At these foci laminin, a basement membrane glycoprotein was specificially localised to intracellular epithelial sites, whereas in more developed areas, laminin was restricted to epithelial basement membranes. Associated with the more mature structures, laminin was also localised to intracellular granules of renomedullary interstitial cells. Thus, renomedullary interstitial cells are present prior to and appear to be actively associated with tubule repositioning in the medulla, establishing themselves as integral to the process of renal development.

Age Factors↗
PubMed1996

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

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.

Animals↗
PubMed1996

Biphasic glomerular hypertrophy in rats administered puromycin aminonucleoside.

Recent evidence suggests that glomerular hypertrophy is a key event in the development of focal and segmental glomerulosclerosis and hyalinosis (FSGS) in humans and in many experimental models of FSGS. The initial aim of the present study was to determine if glomerular hypertrophy occurs in a puromycin aminonucleoside (PAN) model of FSGS, previously considered not to involve glomerular hypertrophy. Upon identifying significant glomerular hypertrophy, our second aim was to determine the contribution of glomerular capillary growth to this hypertrophy. Female Sprague-Dawley rats (approximately 200 g) were administered either PAN (2 mg/100 g body wt) subcutaneously, or an equivalent volume of 0.9% saline at weeks 0, 1, 2, 4, 6, 8 and 10. Tissue was analyzed at weeks 7 and 13. Unbiased stereological methods were used to estimate a range of glomerular parameters. Mean glomerular tuft volume in PAN-treated rats was 48% greater than in saline-treated rats at seven weeks, and 63% greater at 13 weeks. Similar results were found for mean renal corpuscle volume. FSGS was absent at seven weeks and minor at 13 weeks. Two-way analysis of variance indicated: significant effects (P < 0.05 at least) of PAN on capillary length per glomerulus, capillary surface area per glomerulus, capillary diameter and length of capillaries per unit volume of glomerulus; and significant effects of time on capillary diameter, capillary length per unit volume of glomerulus and capillary surface area per unit volume of glomerulus. The mean length of capillaries per glomerulus was 45% greater in PAN-treated rats at week 7 and 22% greater in PAN-treated rats at week 13. Taken together, these results indicate a biphasic pattern of glomerular hypertrophy in this model. In the first phase (to 7 weeks), an increase in capillary length contributes to glomerular hypertrophy. In the second phase (7 to 13 weeks), the continued glomerular enlargement appears more likely to be due to an increase in capillary diameter and/or mesangial matrix expansion.

Analysis of Variance↗
PubMed1994

Immunohistochemical studies of renin-containing cells in the developing sheep kidney.

BACKGROUND: Renin-containing (RC) cells in small ruminant kidneys have been known to be widely distributed along the blood vessels. In the present study, RC cells in developing sheep kidneys were studied to investigate not only the appearance but distribution with the potential physiological significance using immunohistochemical and histoplanimetrical techniques. METHODS: Seven fetal, 12 newborn, and 3 adult metanephric kidneys were used and immunostained by anti-renin antiserum. In the histoplanimetrical analysis, the numerical values of RC cells existing at the walls of 3 major arterial types in the kidneys were calculated. RESULTS: At day 44 of gestation, RC cells were already demonstrated in the walls of renal, interlobar, and afferent vessels, located in the deep cortex and the medulla. In intermediate gestational periods, RC cells were detected throughout the intrarenal arterial trees. In late gestational periods, RC cells expressed in the walls of interlobar/arcuate and interlobular arteries tended to decrease or disappear gradually, while they were distributed predominantly in the afferent glomerular vessels. In newborn lambs, especially days 1 to 3 after birth, increased numbers of RC cells were demonstrated throughout the arterial trees in the kidneys. In older lambs, RC cells located in the interlobar/arcuate arteries and the proximal region of the interlobular arteries decreased in number and gradually disappeared. Some RC cells were still distributed in the distal portion of the interlobular artery even in the adult sheep. CONCLUSIONS: These results suggest that the wide distribution of RC cells in sheep kidney is formed in perinatal life, and that the neuronal regulation is associated with the maintenance of this distribution.

Aging↗
PubMed1994

Reactive oxygen species in puromycin aminonucleoside nephrosis: in vitro studies.

We examined the role of reactive oxygen species (ROS) in puromycin aminonucleoside (PAN)-induced changes to glomerular epithelial cells (GECs) in vitro. Levels of superoxide anion (O2.-), hydrogen peroxide (H2O2) and hydroxyl radical (HO.) were measured in rat kidney-slice cultures containing PAN with or without antioxidants (allopurinol, probucol and alpha-tocopherol/ascorbic acid). GEC morphology was assessed after three days of culture using transmission (TEM) and scanning (SEM) electron microscopy. The effects of hypoxanthine on GEC ultrastructure was also assessed. O2.-, H2O2 and HO. were generated when PAN was added to kidney-slice cultures in Medium 199. TEM morphometry revealed that incubation with PAN (100 micrograms/ml) significantly (P < 0.05 at least) retarded the loss of GEC foot processes normally seen in vitro. When the hydrophobic antioxidants probucol or alpha-tocopherol/ascorbic acid, which scavenged/inhibited generation of O2.-, H2O2 and HO., were added to cultures containing PAN, the effect of PAN on foot processes was abolished. The TEM appearance of GECs now resembled that seen in control cultures. On the other hand, SEM revealed that probucol and alpha-tocopherol/ascorbic acid provided no protection against the changes induced by PAN in GEC cell bodies or major processes. Allopurinol provided no protection against the changes induced by PAN in GEC cell bodies, major processes or foot processes. The addition of hypoxanthine to kidney-slice cultures did not result in the generation of O2.-, H2O2 or HO., or alter GEC ultrastructure. These findings indicate that ROS play a role in PAN-induced alterations to GEC foot process architecture in vitro. However, the xanthine oxidase pathway does not appear to play a major role in generating ROS from PAN in vitro.

Animals↗
PubMed1994

Antioxidants protect podocyte foot processes in puromycin aminonucleoside-treated rats.

Whether a reduction in urinary protein excretion in rats coadministered puromycin aminonucleoside and antioxidants was associated with a reduction in alterations to glomerular epithelial cell (podocyte) ultrastructure was examined. Daily urinary protein excretion was measured in rats that received a single i.v. injection of saline or puromycin aminonucleoside with or without coadministration of antioxidants. The coadministration of alpha-tocopherol/ascorbic acid, dimethyl thiourea, or superoxide dismutase to puromycin aminonucleoside-treated rats reduced proteinuria by approximately 90, 40, and 60%, respectively, over the 18-day period studied. For a second group of rats, daily urinary protein excretion was measured and kidneys were processed for light microscopy and transmission and scanning electron microscopy 4, 5, and 10 days after injection. Transmission electron microscopic morphometric analysis of glomeruli from puromycin aminonucleoside-treated rats coadministered antioxidants revealed significantly reduced foot process effacement on Days, 4, 5, and 10 compared with rats that received puromycin aminonucleoside alone. Thus, at Day 10, puromycin aminonucleoside-treated rats coadministered alpha-tocopherol/ascorbic acid, dimethyl thiourea, or superoxide dismutase contained 90, 74, and 88% (P < 0.01 in all cases) more glomerular epithelial cell filtration slits per unit length of glomerular basement membrane than rats treated with puromycin aminonucleoside alone. In contrast, by scanning electron microscopy, the antioxidants were found to provide no protection against the changes occurring in glomerular epithelial cell bodies and major processes. These results provide further evidence of a role for reactive oxygen species in puromycin aminonucleoside nephrosis and indicate that the antioxidants provide protection against the changes occurring in glomerular epithelial cell foot processes.

Animals↗
PubMed1994

Effects of angiotensin converting enzyme inhibition on glomerular number, juxtaglomerular cell activity and renin content in experimental unilateral hydronephrosis.

OBJECTIVE: To determine the effects of hydronephrosis on glomerular number and juxtaglomerular cell synthetic activity and the protective influence of angiotensin converting enzyme inhibition. DESIGN: A comparison of sham and contralateral kidneys with 8-week ipsilateral ureteral ligated hydronephrotic kidneys in BALB/c mice. Enalapril was administered from 5 weeks in additional sham and hydronephrotic kidney groups. METHODS: Renin and prorenin immunohistochemistry was applied to sections of perfusion-fixed kidneys at the light and electron microscope level. Glomerular number was estimated by a physical disector-fractionator stereological method. An enzyme kinetic renin assay was performed in kidney tissue and plasma. RESULTS: Glomerular number in hydronephrotic kidneys decreased significantly compared with sham and contralateral kidneys. Renin content in hydronephrotic kidneys did not change compared with sham or contralateral kidneys, but the renin content in the glomerulus was significantly greater in hydronephrotic than in contralateral kidneys and similar to in sham kidneys. Contralateral kidneys enlarged significantly and their total renin content decreased significantly compared with hydronephrotic and sham kidneys. Plasma renin was unchanged. Fewer juxtaglomerular cells were labelled for renin and prorenin in contralateral than in hydronephrotic or sham kidneys. Granulopoiesis and exocytotic profiles were markedly greater in hydronephrotic than in contralateral or sham kidneys. Following enalapril, glomerular number was significantly higher in hydronephrotic kidneys and renin content increased proportionally more in contralateral than in hydronephrotic or sham kidneys. CONCLUSION: Hydronephrosis for 8 weeks results in atrophy of 50% of glomeruli and exerts an inhibitory influence on contralateral juxtaglomerular cells while augmenting ipsilateral renin production per remaining glomerulus with maintenance of plasma renin. Enalapril preserves glomeruli and reverses the contralateral inhibitory influence, suggesting an angiotensin-related mechanism.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1993

Scanning and transmission electron-microscopic study of peripolar cells in the newborn lamb kidney.

Scanning and transmission electron microscopy were used to study the ultrastructural characteristics and positions of granulated peripolar cells in newborn lamb kidney. Following tissue fixation by vascular perfusion in situ, the vascular pole region of the glomerulus was exposed for examination by scanning electron microscopy following removal of the glomerular tuft. Peripolar cells were recognized by their surface morphology enabling their quantification and an assessment of the relationship of their position in the renal cortex. The prominent expression of peripolar cells in this species was confirmed. Almost every vascular pole examined revealed peripolar cells (405 out of 407; 99.5%) and thus, throughout the cortex, the distribution of peripolar cells was the same as the distribution of renal corpuscles. Larger, more protruding peripolar cells were observed in the outer cortical renal corpuscles. The numbers of peripolar cells encircling each vascular pole ranged from 1 to 10. There was no correlation between number of granulated peripolar cells at the vascular pole and the position of the renal corpuscle within the renal cortex. As viewed by transmission electron microscopy, organelles of protein synthesis were abundant in the cytoplasm of peripolar cells. Exocytosis of cytoplasmic granules was observed by both scanning and transmission electron microscopy implying that a process of regulative secretion occurs from these cells. The use of ultrastructural techniques has provided evidence supporting the concept that peripolar cells are prominent in the cuff region of each renal corpuscle of the newborn lamb and furthermore that peripolar cells in this species most likely have a secretory function.

Animals↗
PubMed1993

Production of monoclonal antibodies recognising different epitopes present on insulin-like growth factor 1.

Monoclonal antibodies (MAbs) were generated against recombinant human insulin-like growth factor 1 (IGF-1) by fusion of NS-1 myeloma cells with spleen cells from BALB/c X DBA mice immunised with recombinant IGF-1 and synthetic peptide sequences derived from the published amino acid sequence of IGF-1. MAbs were produced that recognised four distinct epitopes including two defined segments of the C and D domains. All MAbs were IgM mouse immunoglobulins. These results indicate the feasibility of producing MAbs to highly conserved proteins.

Amino Acid Sequence↗
PubMed1993

Renin processing in cultured juxtaglomerular cells of the hydronephrotic mouse kidney.

We examined renin processing in cultured juxtaglomerular (JG) cells of the hydronephrotic mouse kidney with immunocytochemical and biochemical techniques. Compared with JG cells in normal kidneys, there was less intense labeling for renin protein in mature granules of cultured JG cells. However, pro-renin labeling of transport vesicles and juvenile granules was maintained, suggesting incomplete passage of pro-renin through intermediate and mature granules. Immunogold evidence of exocytosis of mature granules containing renin protein was present at all stages. Labeling of transport vesicles for pro-renin, together with the absence of exocytosis of pro-renin from juvenile granules, indicated that pro-renin was exclusively released by a constitutive process. Active renin release into supernatants decreased with time, whereas the ratio of total renin to active renin increased, indicating that pro-renin synthesis and release were maintained but that the processing of pro-renin to active renin was interrupted. Angiotensin II inhibited and verapamil stimulated active renin release in culture; neither substance affected pro-renin release. Application of secretagogues that act via intracellular calcium or cAMP resulted in depletion of mature granules and their deformation by myelin figures and vacuoles, findings consistent with an exocytosis from mature granules. The absence of effect of any secretagogues on pro-renin release suggests that these stimulatory mechanisms are exclusively post-Golgi. In cultured JG cells in renal explants, renin vesicular transport and granular exocytosis are maintained but a defect in pro-renin passage from juvenile to intermediate granules is apparent.

Angiotensin II↗
PubMed1993

The glomerular peripolar cell.

The PPC is a distinctive granulated epithelial cell at the vascular pole of the glomerulus. It is present in a wide range of animal species. It is especially prominent in sheep, particularly the newborn lamb. To date, it has been shown that its granules contain albumin, immunoglobulins, neuron-specific enolase and transthyretin. It does not appear to contain renin. The function of the PPC awaits clarification. It has been postulated that it may play a role in the synthesis and secretion of factors involved in modulating renal tubular function.

Animals↗
PubMed1992

Immunohistochemical localization of transthyretin in glomerular peripolar cells of newborn sheep.

Purified transthyretin has been isolated from sheep serum. Antiserum raised against this protein has been used with an indirect immunoperoxidase histochemical technique to identify transthyretin in newborn lamb kidney tissue. Transthyretin was found in proximal tubule cells and in glomerular peripolar cells. Preabsorption studies using purified transthyretin protein indicate that the immunoreactivity of the antiserum is specific to transthyretin.

Animals↗
PubMed1992

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

Immunogold 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 proto- and 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.

Animals↗
PubMed1992

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

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 glycol-methacrylate (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 31,764 +/- 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.

Animals↗
PubMed1990

In vitro effects of puromycin aminonucleoside on the ultrastructure of rat glomerular podocytes.

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 micrograms/ml and 500 micrograms/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 less than 0.001 in all cases). TEM morphometry revealed that incubation with 500 micrograms/ml PAN retarded significantly (p less than 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.

Animals↗
PubMed1990

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

Atrial 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 approximately 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 volume-loaded 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 less than 0.05). A significantly similar difference was found for nephrectomy/volume-loaded animals. There was no significant difference in Vv for right appendage between the control and experimental groups; for left appendage there was a significant increase in Vv to 2.42 +/- 0.09% (p less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

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.

Animals↗