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Notes: 1. We have previously shown that [125I]-endothelin (ET) receptor binding is localized almost exclusively to the fenestrated endothelial cells of glomerular capillaries and peritubular capillaries in the rat kidney following systemic administration of the radioligand in vivo. Because of the lack of specific ET receptor binding in other glomerular and tubular structures following in vivo labelling, we undertook further studies, using electron microscopic autoradiography and ET receptor subtype selective ligands, to investigate whether other renal components also contain ET receptor binding and, if so, to determine the cellular localization of the ET receptor subtypes, ETA and ETB, following in vitro labelling.2. At the electron microscopic level, ET binding sites were localized primarily to the fenestrated endothelium of glomerular and peritubular capillaries of the cortex, inner stripe of the outer medulla and the inner medulla. ET binding sites also occurred overlying renomedullary interstitial cells (RMIC) of the inner medulla.3. The ETB receptor selective agonist, sarafotoxin 6c (S6c), abolished ET binding in the vascular endothelium throughout the kidney, while the ETA receptor selective antagonist, BQ123, was without effect. Both BQ123 and S6c partially inhibited the binding in the RMIC of the inner medulla.4. These results indicate that ET receptor binding in the fenestrated endothelium in the glomerular capillaries and peritubular capillaries belongs mainly to the ETB subtype, whereas both ETA and ETB subtypes are present in the RMIC.

Notes: 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.2. The effects of ACE inhibition were investigated at a stage of development before the renal outer medulla is fully established.3. Sprague-Dawley rat pups were given daily i.p. injections of either enalapril or saline from days 3–10. At day 11, kidneys were perfusion-fixed for either electron microscopy or immunocytochemistry. Sections were incubated in proliferating cell nuclear antigen (PCNA) antisera and the avidin-biotin immunoperoxidase method was used to detect an immunoreactive product, indicative of proliferating cells.4. Following enalapril treatment, the normal structural arrangement of the outer medulla was disrupted compared with controls. Cell proliferation (PCNA-positive cells) in the medullary rays was reduced in enalapril-treated kidneys compared with control kidneys.5. Thus, angiotensin II appears to be essential for normal tubular and vascular growth in postnatal renal development in the rat.

Notes: 1. The earliest form of the kidney, the pronephros, does not really occur in the ovine embryo; instead, a giant glomerulus forms at the anterior end of the mesonephros.2. In the sheep, the mesonephros is present from 11-38% of total gestation (150 days) and produces a dilute urine, as well as expressing the genes for erythropoietin, renin, angioten-sinogen, angiotensin-converting enzyme and the angiotensin II (AngII) receptors AT1 and AT2.3. The ovine metanephros begins to develop at 18% of gestation and nephrogenesis is complete several weeks before birth. AH components of the renin-angiotensin system (RAS) are expressed from at least 27% of gestation.4. Both AT1 and AT2 receptors are expressed by the adrenocortical cells early in gestation but, at mid-gestation, exogenous AngII does not stimulate aldosterone secretion in vivo.5. Preliminary results suggest that Angll has important roles in renal development in the ovine foetus but the role(s), if any, in adrenal development, remains to be investigated.

Notes: 1. Cultured renomedullary interstitial cells (RMIC) isolated from 4-week-old Sprague-Dawley rat kidneys possess ETA receptors, as identified by reverse transcription–polymerase chain reaction (RT-PCR).2. Treatment with endothelin (ET)-1 (10−6 mol/L) increases the intracellular inositol 1,4,5-trisphosphate concentrations within 10 s and intracellular calcium concentrations after 7 s.3. Endothelin-1 (10−7 and 10−10 mol/L) induced increases in intracellular cAMP concentrations, but only in the presence of Nω-nitro-L-arginine, a nitric oxide synthase (NOS) inhibitor. Addition of ET-1 (10−10 mol/L) to the RMIC culture led to increases in intracellular cGMP concentrations through activation of NOS.4. In the presence of ET-1 (10−7 and 10−10 mol/L) and during NOS inhibition, RMIC responded with increased cell proliferation and extracellular matrix (ECM) synthesis. These responses were abolished by BQ-123 (10−6 mol/L), suggesting mediation via the ETA receptor subtype. The proliferative effect of ET-1 was also abolished by atrial natriuretic peptide (10−6 mol/L).5. The present study provides evidence that binding of ET-1 to ETA receptors on RMIC activates several intracellular second messenger systems that mediate cell proliferation and ECM synthesis.6. These results also highlight an important interaction between ET-1 and nitric oxide in the control of RMIC function.

Notes: Summary: Quantitative methods are frequently used to analyse the structure of renal glomeruli. However, on most occasions, measurements are made on glomerular profiles (the two-dimensional samples of glomeruli seen in histological sections), and provide little or no information about the structure of whole, three-dimensional glomeruli. Stereology is the discipline concerned with the quantitative analysis of three-dimensional structures. With stereology one can estimate the total number of glomeruli in kidneys, as well as mean glomerular volume, the number of cells in glomeruli, and the length and surface area of glomerular capillaries. In addition to providing a means for detecting structural differences between glomeruli from different specimens, stereology provides quantitative structural information that can be correlated with quantitative physiological, biochemical and molecular data. Over the past decade we have witnessed the development of a new generation of unbiased, cost-efficient stereological methods that are ideally suited to analysing glomeruli. Some of these methods are introduced in this review, and then three recent studies from our laboratories that successfully utilized these methods are described. These studies concerned hypertension, kidney development, and the pathogenesis of focal and segmental glomerulosclerosis.

Notes: Summary: Fibroblast growth factors (FGF) regulate cell proliferation, migration, differentiation and angiogenesis during morphogenesis in many different tissues. Recent evidence indicates that exogenous FGF-2 stimulates mesenchymal condensation in cultured rat metanephroi, a crucial epithelial-mesenchymal induction event in the developing nephron. the aim of the present investigation was to determine the in vivo distribution of FGF-1 and FGF-2 in developing rat metanephroi at embryonic days 14, 15, 16, 18 and 20. Avidin-biotin enhanced indirect immunohistochemistry was used to demonstrate that both FGF-1 and FGF-2 were co-localized in metanephroi at all ages studied. High levels of FGF-1 and FGF-2 were present in ureteric bud branches and in developing distal tubules. Fibroblast growth factor-1 and FGF-2 were colocalized in developing nephron elements, from vesicles to S-shaped bodies, and in the mesangium of capillary loop and maturing stage glomeruli. Both growth factors were present in the mesenchyme of the nephrogenic zone and in the interstitium of the developing cortex. However, immunostaining for FGF was not evident in mesenchymal condensates, endothelial cells, medullary interstitial cells, or in the thin undifferentiated epithelium of the immature loop of Henle. These findings indicate that the expression of both FGF-1 and FGF-2 is tightly regulated in the embryonic kidney and suggest a role for these molecules in kidney development.

Notes: 〈list xml:id="l1" style="custom"〉1There is strong evidence for a renal basis to the development of hypertension in the spontaneously hypertensive rat (SHR). Alterations of the SHR renal vasculature, including the glomerulus, may be involved in the initiation and maintenance of hypertension in this animal model.2The arterial walls of pre-glomerular vessels of the SHR are hypertrophied compared with WKY vessels. Unlike other vascular beds in the SHR, this hypertrophy is independent of angiotensin II (AngII).3Glomerular number and volume are similar between SHR and the normotensive Wistar-Kyoto (WKY) rats. These results provide no support for the theory that a reduced filtration surface area within the kidneys of the SHR contributes to the elevated blood pressure in these animals.4Intrarenal hypertrophy may have similar haemodynamic consequences to clipping of the main renal artery, as in Goldblatt hypertension. Further analysis of the role of pre-glomerular arterial hypertrophy is warranted to determine its involvement in the initiation and maintenance of hypertension in the SHR.

Notes: Summary: The transforming growth factor-β (TGF-β) family of growth factors regulates cell proliferation, differentiation, extracellular matrix synthesis and angiogenesis in many developing tissues. Transforming growth factor-β1 was recently shown to affect the branching of ureteric epithelium and nephron formation in cultured rat metanephroi. As the TGF-β type II receptor is specific for the TGF-β family, the present study used in situ hybridization to localize mRNA for this receptor in metanephroi from Sprague-Dawley rat embryos. Transforming growth factor-β type II receptor mRNA was located in ureteric duct epithelium, undifferentiated mesenchymal cells in the nephrogenic zone, vesicles, comma-shaped bodies and S-shaped bodies. In some S-shaped bodies, TGF-β type II receptor mRNA was not expressed in the lower limb, which subsequently forms the renal corpuscle. Expression was not observed in capillary loop stage glomeruli and maturing glomeruli, or in proximal tubules and interstitial cells. In adult rat kidney, TGF-β type II receptor mRNA was expressed in cortical collecting ducts and distal tubules but not in glomeruli or proximal tubules. These findings demonstrate that the prominent expression of TGF-β type II receptor mRNA decreases as glomeruli and tubules develop. Expression then remains undetectable in adult glomeruli and proximal tubules. the developmentally-regulated expression of this receptor suggests a key role in glomerular and nephron development.

Notes: Summary: Angirotensin II (AII) is a powerful humoral regulator of body fluid and electrolyte balance and arterial blood pressure. In the kidney, All influences renal haemodynamics and proximal tubular reabsorption of sodium through activation of All that mediate complex signal transduction pathways. Angiotensin II is also implicated in the pathophysiological process of some progressive renal diseases. Pharmacological characterization and molecular cloning of All receptor reveals at least two major subtypes of All receptors, AT1 and AT2, in the kidney and other tissues. the AT1 receptor cDNA encodes a 359 amino acid protein with structure typical of seven transmembrane G-protein coupled receptors. Two isoforms of AT1 receptor, AT1A and AT1B, are known in rodents, but probably only one occurs in other mammals including humans. the AT2 receptor cDNA, a 363 amino acid protein, shares only 32% identical amino acid residues with AT1 receptor, although it also has a seven transmembrane domain topology. In adult mammalian kidneys, AT1 receptors predominate in the glomerular mesangium, proximal tubular epithelium, renomedullary interstitial cells in the inner stripe of the outer medulla and large preglomerular vessels except those in human and monkey where AT2 receptors predominate. By contrast, in foetal kidneys, AT2 receptors are the major subtype; however, this shows dramatic regulation during development. Physiological studies using AT1 selective antagonists show that the known actions of All on renal haemodynamics, glomerular filtration, and tubular sodium and water transport are mediated by this subtype of All receptors. In addition, AT1 receptors also mediate hypertrophic and mitogenic actions of All on cultured glomerular mesangial cells and proximal tubular epithelial cells, and on extra-cellular matrix accumulation in animal models of progressive renal diseases. By contrast, blockade of AT2 receptors has no effect on renal haemodynamics, tubular sodium reabsorption or growth properties of All. Overall, All exerts multiple actions in the kidney by interacting with different subtypes of All receptors located on multiple cellular sites.

Notes: 1. Recent physiological experiments have established that increasing the perfusion pressure of the kidney causes the release of a vasodepressor substance from the renal medulla.2. The substance is not a platelet activating factor, a prostaglandin or nitric oxide and the vasodepressor response to increased renal perfusion pressure is not due simply to inhibition of renin release.3. The mechanisms by which the renomedullary vasodepressor substance lowers arterial pressure remain to be determined. Sympathoinhibition may account for part of the response, but the hypotension still occurs in autonomic ganglion blocked animals.4. The source of the substance appears to be the renomedullary interstitial cells, though the control of the production and release of the substance remain to be determined.5. The substance may be a lipid but it is yet to be fully isolated and identified.6. The threshold for release of the substance appears to be close to normal resting arterial blood pressure.7. Despite strong evidence that the renal medulla releases a vasodepressor hormone in response to increased renal perfusion pressure, much is still to be determined regarding the physiology of this hormone and its involvement in the aetiology of hypertension.