Daine Alcorn

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.

Presence of angiotensin II AT2 receptor binding sites in the adventitia of human kidney vasculature.

1 Angiotensin II (AngII) receptor subtypes in adult human kidney were pharmacologically characterized by in vitro autoradiography using the AngII receptor subtype‐selective antagonists, losartan and PD 123319, and the sensitivity to the reducing agent, dithiothreitol. 2 High densities of AngII AT1 receptor binding occur in the glomeruli and the inner stripe of the outer medulla, while a moderate AT1 receptor binding is localized in the proximal convoluted tubules. 3 AT2 receptor binding is observed predominantly in the intrarenal large blood vessels, including the arcuate, inter‐ and intra‐lobular arteries, and in the renal capsule. 4 In the major renal artery, AT1 receptor binding is abundant in the media and adventitia, while AT2 receptor binding is observed mainly in the adventitia. 5 At the light microscopic level using emulsion autoradiography, AT1 receptors are localized in the glomeruli and juxtaglomerular apparatus, as expected. However, in larger renal blood vessels, including the arcuate arteries, inter‐ and intra‐lobular arteries, intense AT2 receptor labelling occurs primarily in the adventitia, while the endothelium and vascular smooth muscle layers contain only low levels of AngII receptor binding. 6 These results indicate that the adult human kidney displays two pharmacologically distinct AngII receptor subtypes, with AT1 predominating in the glomeruli, juxtaglomerular apparatus, proximal tubules and the inner stripe of the outer medulla, while AT2 predominates in the adventitia of the arcuate and interlobular arteries and the renal capsule. The functional significance of AT2 receptor binding sites in the adventitia of adult human kidney vessels remains to be elucidated.

Enalapril Does Not Prevent Renal Arterial Hypertrophy in Spontaneously Hypertensive Rats

Angiotensin-converting enzyme inhibitors prevent the development of vessel wall hypertrophy in some vascular beds in spontaneously hypertensive rats (SHR), but their effects on hypertrophy of renal arterial vessels have not been studied. We therefore used stereological techniques to study wall and lumen dimensions of the interlobular (cortical radial) and arcuate arteries in the kidneys of SHR (n = 7), SHR treated from 4 to 10 weeks of age with enalapril (25 to 30 mg/kg per day; SHR-E, n = 7), and Wistar-Kyoto rats (WKY, n = 7). All kidneys were perfusion-fixed at 10 weeks. Systolic blood pressure was 199 +/- 9, 139 +/- 11, and 156 +/- 8 mm Hg in the SHR, SHR-E, and WKY groups, respectively. For the interlobular arteries, the volume density of artery wall, wall-to-lumen ratio, and wall thickness in the untreated SHR were significantly greater than in the WKY (0.84 +/- 0.09 versus 0.69 +/- 0.07 x 10(-3), 0.75 +/- 0.20 versus 0.53 +/- 0.08, and 13.6 +/- 3.3 versus 10.6 +/- 0.8 microns, respectively), but values in the SHR-E were similar to those in the untreated SHR (1.10 +/- 0.20 x 10(-3), 0.88 +/- 0.22, and 14.0 +/- 2.6 microns, respectively). For the arcuate arteries, wall thickness and volume density were significantly greater in SHR than WKY (17.3 +/- 3.0 versus 13.9 +/- 1.7 microns and 1.63 +/- 0.51 versus 1.14 +/- 0.27 x 10(-3), respectively), and values in the SHR-E (15.7 +/- 1.7 microns and 1.69 +/- 0.50 x 10(-3), respectively) were not significantly different from those in SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of the renal interstitium.

Abstract The kidney is derived from two tissue sources and develops through a series of mesenchymal-epithelial transitions and epithelial-mesenchymal interactions to form an epithelial tubular organ embedded in an interstitium derived from mesenchyme. The primary interstitium of the embryonic kidney undergoes significant differentiation to form the adult counterpart whose diverse cells have structural and functional characteristics that relate to their local milieu. Whether the adult interstitial cells retain the capacity to transform to other cell types and thus play a role in pathophysiological conditions appears more and more likely as the plasticity of cells becomes apparent. Besides forming the adult interstitium, the primary interstitium is active in metanephric development, with specific roles in nephron growth and collecting duct growth and arborization. Interruptions to the development of the interstitium or amelioration of its developmental capacity result in severely disrupted kidneys. The development of the renal interstitium is an essential component in the process of renal genesis.

Glomerular dimensions in spontaneously hypertensive rats: effects of AT1 antagonism.

Objective A reduction in glomerular number and/or size has been implicated in the development of hypertension. This study investigated whether differences in glomerular number and/or size occur during the development of hypertension in the spontaneously hypertensive rat (SHR) and whether angiotensin II is responsible for any glomerular differences. Methods SHR (n=6) and Wistar–Kyoto (WKY) rats (n=6) were administered the angiotensin II type I receptor antagonist TCV-116 from 4 to 10 weeks of age. At 10 weeks of age, the kidneys from these rats and those from untreated SHR (n=6) and WKY rats (n=6) controls were perfusion fixed at physiological pressures and analysed using unbiased stereological techniques. Results There were no significant differences in glomerular number, glomerular volume or total glomerular volume between SHR and WKY rats. Treatment of SHR with TCV-116 significantly lowered systolic blood pressure but had no significant effect on glomerular number or volume or total glomerular volume. Treatment of WKY rats with TCV-116 reduced systolic blood pressure, body weight, glomerular volume and total glomerular volume; however, total glomerular volume per body weight of treated WKY rats was not significantly different from that of untreated WKY rats. Conclusion There were no differences in glomerular number or volume in SHR compared with WKY rats at 10 weeks of age. We therefore conclude that glomerular changes are not responsible for the development of hypertension in SHR. Angiotensin II, via the type 1 receptor, does not contribute to glomerular growth during the development of hypertension in the SHR.

In vivo occupancy of angiotensin II subtype 1 receptors in rat renal medullary interstitial cells.

Angiotensin II receptor binding sites in type 1 interstitial cells in the inner stripe of the outer medulla are readily labeled in vitro by the radioligand but not in vivo after systemic radioligand administration. In anesthetized rats, we investigated if reduced vascular delivery due to angiotensin II-induced renal vasoconstriction or, alternatively, prior occupancy of these sites by endogenous angiotensins modulates angiotensin II subtype 1 receptor binding to renal medullary interstitial cells in vivo using electron microscopic autoradiography. Using 125I-angiotensin II, administered systemically, as a radioligand, binding in control rats occurred predominantly in the glomeruli and proximal tubules, while only low binding was observed in the inner stripe of the outer medulla. Pretreatment of rats with unlabeled [Sar1,Ile8]angiotensin II or with the angiotensin II subtype 1 receptor antagonist losartan before receiving the radioligand completely abolished binding to all sites. Renal vasodilatation induced by sodium nitroprusside or use of the radiolabeled antagonist analogue 125I-[Sar1,Ile8]angiotensin II did not alter binding to the inner stripe. In contrast, chronic salt loading or inhibition of angiotensin-converting enzyme by perindopril significantly increased binding not only to the cortical sites but also to the sites in the inner stripe of the outer medulla. Electron microscopic autoradiographs of the inner stripe detected binding in the interstitial cells only in rats treated with chronic salt loading or perindopril. These results suggest that endogenous angiotensins may modulate binding of circulating angiotensin II to the interstitial cells in vivo, and these angiotensin II receptor-bearing cells are more likely to be more responsive to interstitial angiotensin II than to the circulating hormone.

Effect of converting enzyme inhibition on renin synthesis and secretion in mice.

We have investigated the relative importance of renal renin stores and de novo synthesis during stimulation of renin secretion and the role of transcription and posttranscriptional factors in providing increased synthesis of renin. When enalapril was administered to previously untreated mice, plasma renin concentration increased 40-fold within 1.5 hours, and remained at a high level for the 8 days of the experiment. Renal renin decreased by 82% after 24 hours and thereafter increased to levels higher than controls. Calculations of renin turnover, based on data for the rate of metabolism of renin in plasma, indicated that most of the renin released in the first 24 hours could be accounted for by the decrease in renal renin stores, indicating that de novo synthesis played only a minor role. After 24 hours, however, when both plasma renin concentration and renal renin increased, the calculated rate of renin synthesis increased to nearly 40 times the rate in controls. When enalapril was administered to mice that had been depleted of plasma and renal renin by chronic sodium loading, plasma renin concentration increased markedly within 1.5 hours, but to only half the level achieved in the previously untreated mice. No decrease in renal renin occurred, suggesting that the renal renin remaining after chronic sodium loading was not available for release. Renal renin messenger RNA increased 4.5-fold after 6 hours, and after 8 days had increased to 5.0 times the level at day 0. The increase in calculated rate of renin synthesis was maximal between 5 and 8 days, when it was 54 times greater than at day 0. During enalapril treatment, there were marked increases in the granulation of the juxtaglomenilar cells and in the amount of rough endoplasmic reticulum and Golgi apparatus they contained. These results suggest that posttranscriptional factors play a major role in determining the rate of renin synthesis.

EVIDENCE FOR A RENOMEDULLARY VASODEPRESSOR HORMONE

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.

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.

CELLULAR LOCALIZATION OF ENDOTHELIN RECEPTOR SUBTYPES IN THE RAT KIDNEY FOLLOWING IN VITRO LABELLING

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.