Quantitative changes in rat renin secretory granules after acute and chronic stimulation of the renin system

In order to study the cellular mode of renin secretion, stereological methods were used to estimate number and volume of rat renin secretory granules during stimulation of the renin system. An acute decrease in renal perfusion pressure to 40 mmHg for 5 min increased plasma renin concentration (PRC) twofold, but did not significantly change the number of renin granules per arteriole or the renin-containing volume of the arteriole. Chronic stimulation was achieved by a combination of low-salt diet and inhibition of angiotensin-converting enzyme (ACE) for 14 days, and resulted in a 36-fold increase in PRC, a 20-fold increase in the number of granules per arteriole, and a 17-fold increase in the arteriolar volume that contained renin. An acute decrease in renal perfusion pressure to 40 mmHg for 5 min in the chronically stimulated rats increased PRC further (1.6-fold), and significantly reduced the number of granules per arteriole by 4000 (45% reduction), but did not change the renin-containing arteriolar volume significantly. The average renin granule size was 0.35 μm³ with no significant differences among the groups. We conclude that recruited granular cells contribute significantly to renin release, and that all granular cells along the arteriole participate in secretory responses. The reduced number of renin granules after acute stimulation is compatible with exocytosis as the dominating mechanism of renin release.     

Renal microvasculature of the black bear (Ursus americanus)

This study of the Black bear (Ursus americanus) was undertaken to provide basic information related to structural modifications in the renal microvasculature that might provide insight into the drastic alteration in renal urinary output that occurs during winter sleep. Vascular casts, as well as light microscopy and scanning electron microscopy, were used to study the vascular components of the juxtaglomerular complex and related vessels. Histologically, arterial cushions were readily identified at the origin of the afferent arterioles. In the area of the juxtaglomerular complex, the wall of the afferent arteriole appeared to be highly modified. The smooth muscle cells at this site demonstrated a change in morphology and orientation, and the diameter of the arteriole was altered. The pattern of the vascular casts at the origin of the afferent arteriole varied from that portion at the glomerulus, suggesting a modification of the vascular wall near the renal corpuscle. Although the morphology of the renal microvasculature of the Black bear is similar to that of other mammals in some aspects, it is dissimilar to that of other carnivores and of the human kidney in that there are structural modifications of the afferent arteriole that may contribute to a reduction of blood flow to the nephron during winter sleep.     

Myosin content and vasoconstrictive ability of the proximal and distal (renin-positive) segments of the preglomerular arteriole

Summary The PAP-technique and antibodies to myosin were used to demonstrate the prerequisites for vasoconstriction in the juxtaglomerular part of the preglomerular arteriole as compared with its proximal segment in rats and mice. In contrast with the myosin-positive/renin-negative proximal part of the afferent arteriole no myosin-like activity could be demonstrated in its distal, renin-positive part. In accordance, no thick myofilaments were found in fully differentiated juxtaglomerular epithelioid cells replete with mature secretory granules. Stimulation of the renin-angiotensin system was followed by an increase of the reninpositive/myosin-negative portions of the preglomerular arteriole. Marked interspecies and internephron variations in the length of this vessel segment under control and stimulated conditions were observed.The juxtaglomerular part of the preglomerular arteriole close to the macula densa seems therefore to have only limited capabilities for vasoconstriction. This finding may be of importance regarding the tubulo-glomerular feedback, a mechanism allegedly triggered by the so-called macula densa-signal. It is suggested that this non-contractile segment of the afferent arteriole may represent the renal vascular receptor responsible for the increase of renin secretion during pressure reduction.Unlike the afferent arterioles, most of the efferent arterioles showed the highest level of their weak but distinct myosin-like immunoreactivity in the juxtaglomerular region, indicating some efferent juxtaglomerular vasoconstrictive ability.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg     

Angiotensin II in epitheloid (renin containing) cells of rat kidney

The PAP-method was used for immunocytochemical investigations with antisera against angiotensin (ang) I, ang II and renin in kidneys of rats and mice. In 14 rats, ang II was found in the media of the afferent arteriole - both in the region of the JGA and upstream until the interlobular artery. Serial sections alternately reacted for ang II and renin revealed that the octapeptide is contained in the well known renin positive epitheloid cells of the afferent arteriole and, beyond that, together with renin probably in the same "specific" granules. Fixation conditions were critical for the visualization of immunoreactivity With ang I antisera, comparable in terms of titer and affinity to the ang II antisera, specific immunoreactivity could not be found in the kidneys of rats. With horse radish peroxidase and ferritin as tracers it could be shown that the epitheloid cells of the JGA have the ability to pinocytize and incorporate macromolecules into their granules. It is suggested that ang II is taken up by these cells through the same route, Intracellular generation of ang II appears unlikely as an explanation. Functionally the selective uptake of ang II by epitheloid cells might be a specific process, possibly connected with the negative feedback of the octapeptide on renin secretion. Negative results in mice may be explained by a small uptake or more rapid degradation of ang II by the epitheloid cells.     

Insulin downregulates neonatal brain insulin receptors

Using cell cultures rich in renal juxtaglomerular cells we found that a change of the intracellular c-AMP concentration is not a prerequisite for an alteration of the renin secretion rate. Modulators of renin secretion including activators of the adenylate cyclase, however, altered the calcium permeability of the cellular plasma membrane in a way that stimulators of renin secretion lowered the calcium permeability and vice versa. Our results suggest that renin secretion is controlled by the intracellular calcium concentration and not by c-AMP. We postulate that modulators of renin secretion act by changing the calcium permeability of the cell membrane.     

Autoregulation and conduction of vasomotor responses in a mathematical model of the rat afferent arteriole

We have formulated a mathematical model for the rat afferent arteriole (AA). Our model consists of a series of arteriolar smooth muscle cells and endothelial cells, each of which represents ion transport, cell membrane potential, and gap junction coupling. Cellular contraction and wall mechanics are also represented for the smooth muscle cells. Blood flow through the AA lumen is described by Poiseuille flow. The AA model's representation of the myogenic response is based on the hypothesis that changes in hydrostatic pressure induce changes in the activity of nonselective cation channels. The resulting changes in membrane potential then affect calcium influx through changes in the activity of the voltage-gated calcium channels, so that vessel diameter decreases with increasing pressure values. With this configuration, the model AA maintains roughly stable renal blood flow within a physiologic range of blood flow pressure. Model simulation of vasoconstriction initiated from local stimulation also agrees well with findings in the experimental literature, notably those of Steinhausen et al. (Steinhausen M, Endlich K, Nobiling R, Rarekh N, Schütt F. J Physiol 505: 493-501, 1997), which indicated that conduction of vasoconstrictive response decays more rapidly in the upstream flow direction than downstream. The model can be incorporated into models of integrated renal hemodynamic regulation.     

Myoendothelial contacts in glomerular arterioles and in renal interlobular arteries of rat,mouse and Tupaia belangeri

Summary Cell contacts between elements of the tunica media and the intima in the afferent and efferent glomerular arteriole and in the interlobular artery were studied and evaluated semiquantitatively in thin sections of rat and mouse kidney.In the afferent arterioles, including their juxtaglomerular portion, contacts were seen between endothelial and smooth muscle cells, and between endothelial and granulated (renin producing) cells. The form of these musculoendothelial contacts varied from simple appositions of perikarya and cell processes to extensive club-shaped indentations of endothelial cells into media cells (common) or media cells into endothelial cells (rare). Most of these cell contacts seem to contain myoendothelial gap junctions. Fewer, mostly club-shaped myoendothelial contacts were found in the interlobular arteries of rats and mice than in their afferent arterioles. Simple membrane appositions predominated among the numerous myoendothelial contacts of efferent arterioles. Similar results (without quantitative analysis) were obtained in the kidney of Tupaia belangeri. The myoendothelial contacts may allow the detection and propagation of mechanical (autoregulatory) and humoral stimuli.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Control and Modulation of Fluid Flow in the Rat Kidney

We have developed a mathematical model of the rat’s renal hemodynamics in the nephron level, and used that model to study flow control and signal transduction in the rat kidney. The model represents an afferent arteriole, glomerular filtration, and a segment of a short-loop nephron. The model afferent arteriole is myogenically active and represents smooth muscle membrane potential and electrical coupling. The myogenic mechanism is based on the assumption that the activity of nonselective cation channels is shifted by changes in transmural pressure, such that elevation in pressure induces vasoconstriction, which increases resistance to blood flow. From the afferent arteriole’s fluid delivery output, glomerular filtration rate is computed, based on conservation of plasma and plasma protein. Chloride concentration is then computed along the renal tubule based on solute conservation that represents water reabsorption along the proximal tubule and the water-permeable segment of the descending limb, and chloride fluxes driven by passive diffusion and active transport. The model’s autoregulatory response is predicted to maintain stable renal blood flow within a physiologic range of blood pressure values. Power spectra associated with time series predicted by the model reveal a prominent fundamental peak at ～165 mHz arising from the afferent arteriole’s spontaneous vasomotion. Periodic external forcings interact with vasomotion to introduce heterodynes into the power spectra, significantly increasing their complexity.     

Estimation of the number of angiotensin II AT1 receptors in rat kidney afferent and efferent arterioles

OBJECTIVE: To examine the effects of the renin-angiotensin system (RAS) on renal arterioles to determine the association between the distribution of angiotensin II AT1 receptors and the morphologic and physiologic heterogeneity of renal arterioles. STUDY DESIGN: To estimate the number of angiotensin II AT1 receptors along the length of the arterioles and per arteriole, we combined immunoelectron microscopy with stereology. RESULTS: The number of AT1 receptor molecules was significantly lower in the renin-positive smooth muscle cells (SMCs) than in the renin-negative SMCs of the afferent and efferent arterioles. There were no significant differences along and between the afferent and efferent arterioles in relative number of AT1 receptors of endothelial cells or SMCs. CONCLUSION: Our results suggest that the heterogeneous activity of angiotensin II in SMCs and the different permeabilities of the endothelium along the afferent arterioles are probably not controlled directly by angiotensin II AT1 receptors. However, the activity of the RAS is possibly involved in the significantly reduced number of receptors in renin-granulated cells. An understanding of how the number of AT1 receptors on the SMC surface is controlled may furnish a new path for pharmacologically changing RAS activity on SMCs.     

The effect of elevated extracellular glucose on adherens junction proteins in cultured rat heart endothelial cells

Vascular permeability is a proof of vascular endothelial cell dysfunction induced by diabetes. Vascular permeability is directly related to the width of intercellular endothelial cells junctions, which may become permeable to macromolecules as a result of a change in endothelial cell shape. To determine the role of hyperglycemia in endothelial cell shape, the study examined the effect of high concentrations of glucose on the shape of cultured rat heart endothelial cells. This result indicated that the high-glucose-induced changes in the morphology of endothelial cells, via the glucose-mediated reorganization of F-actin. In endothelial cells, the actin cytoskeleton is tethered to the zonula adherens and focal adhesions, which mediate cell-cell and cell-matrix interactions respectively. The present study demonstrated that the high-glucose-induced changes in the actin-binding protein such as filamin, zonula adherens proteins such as alpha-, beta-, and gamma-catenin, focal adhesions proteins such as focal adhesion kinase, paxillin, and tyrosine phosphorylation of paxillin. It appears that differences in expression of adherens junctions molecules on rat heart endothelial cells in response to high glucose reflect endothelial glucose toxicity, which may also induce endothelial dysfunction in diabetes.     

Angiotensin II in epitheloid (Renin containing) cells of rat kidney

Summary The PAP-method was used for immunocytochemical investigations with antisera against angiotensin (ang) I, ang II and renin in kidneys of rats and mice. In 14 rats, ang II was found in the media of the afferent arteriole — both in the region of the JGA and upstream until the interlobular artery. Serial sections alternately reacted for ang II and renin revealed that the octapeptide is contained in the well known renin positive epitheloid cells of the afferent arteriole and, beyond that, together with renin probably in the same specific granules. Fixation conditions were critical for the visualization of immunoreactivity. With ang I antisera, comparable in terms of titer and affinity to the ang II antisera, specific immunoreactivity could not be found in the kidneys of rats. With horse radish peroxidase and ferritin as tracers it could be shown that the epitheloid cells of the JGA have the ability to pinocytize and incorporate macromolecules into their granules. It is suggested that ang II is taken up by these cells through the same route. Intracellular generation of ang II appears unlikely as an explanation. Functionally the selective uptake of ang II by epitheloid cells might be a specific process, possibly connected with the negative feedback of the octapeptide on renin secretion. Negative results in mice may be explained by a small uptake or more rapid degradation of ang II by the epitheloid cells.These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System. A first brief report of the results was given at the Rottach-Egern Satellite Symposium of the VIIth International Congress on Nephrology: The Juxtaglomerular Apparatus and the Tubulo-Glomerular Feedback Mechanism — Morphology, Biochemistry and Function, June 3 to 5, 1981     

Is the renin-angiotensin system involved in urinary concentration mechanisms?

Renin release elicited by i.v. injection of loop-diuretics was used to study the effects of angiotensin II (AII) on intrarenal hemodynamics. The vasoconstrictive action of intrarenally synthesized AII predominates in the efferent glomerular arteriole. Such a vasoconstrictive effect could affect blood flow in the vasa recta which stem from efferent arterioles of juxtamedullary glomeruli. Renin secretion and renal inner medullary blood flow (tissue clearance of 133Xe) were simultaneously measured before and after frusemide-induced renin release. The relationship between renin secretion and renal inner medullary blood flow was inverse. Changes in renal medullary blood flow may be physiological determinants of medullary osmolality and renal concentration ability. The intrarenal role of AII in urinary concentration recovery after frusemide was examined. Inhibition of renin release by propranolol or AII-blockade (by saralasin or Hoe 409) delayed recovery of urinary osmolality. In the conscious rat, propranolol slowed down recovery of the cortico-papillary gradient for sodium. Its vasoconstrictive action on the efferent glomerular arteriole might enable the renin-angiotensin system to participate in the control of renal excretion of salt and water.     

Immunocytochemical demonstration of renin in the mouse and rat kidney after adrenalectomy

Summary Bilateral adrenalectomy in the mouse and rat is followed by a proliferation of renin-producing cells in the kidney, especially in the terminal segments of the afferent arteriole and in the interlobular arteries. The affected cells become larger, and their immune reaction for renin is decreased.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)Dedicated to Prof. Dr. D. Wittekind, Department of Anatomy, University of Freiburg, on the occasion of his 60th birthday     

Microvascular development: learning from pancreatic islets

Microvascular development is determined by the interplay between tissue cells and microvascular endothelial cells. Because the pancreatic islet is an organ composed mainly of endothelial and endocrine cells, it represents a good model tissue for studying microvascular development in the context of a tissue. In this review, we will describe the special morphology of islet capillaries and its role in the physiologic function of islets: secretion of insulin in response to blood glucose levels. We will speculate on how islet-secreted VEGF-A generates a permeable endothelium that allows insulin to pass quickly into the blood stream. In addition, we speculate on how endothelial cells might form a capillary lumen within the islets. At the end, we look at the islet microvasculature from a medical point of view, thus describing its critical role during type I diabetes and islet transplantation.     

Correlation between juxtaglomerular index, kidney renin content and plasma renin concentration in the rat.

The correlation between juxtaglomerular index, kidney renin content, and plasma renin concentration has been investigated in rats. The results indicate that renin exists in two forms. When determining the renin content of the kidney, the renin actually present in the modified smooth muscle cells of the juxtaglomerular apparatus is measured; this is called bound renin. The amount of bound renin is derived from the total of granular and subgranular renin in the modified smooth muscle cells. Since JGI and KRCont show a significant positive correlation in untreated adult rats, it is assumed that in such animals the ratio of granular and subgranular renin is constant. Since no correlation could be demonstrated between kidney renin content and PRC in untreated adult rats, and JGI and KRCont did not change parallel with the increase of PRC in numerous experimental conditions, it is assumed that part of the renin synthetized in the JG cells is secreted directly, without passing the process of condensation into membrane bound granules. This mobile renin does not significantly affect the renin content and the JGI of the kidney. Under physiological circumstances, most of the produced renin seems to mature to granules in the modified smooth muscle cells before being secreted. When renin production and release increased, maturation to granules may be inhibited, a significant part of the produced renin released by direct secretion, and the subgranular, immature renin may also be secreted.     

Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occluden 1. A potential mechanism for vascular permeability in diabetic retinopathy and tumors.

Vascular endothelial growth factor (VEGF) may have a physiologic role in regulating vessel permeability and contributes to the pathophysiology of diabetic retinopathy as well as tumor development. We set out to ascertain the mechanism by which VEGF regulates paracellular permeability in rats. Intra-ocular injection of VEGF caused a post-translational modification of occludin as determined by a gel shift from 60 to 62 kDa. This event began by 15 min post-injection and was maximal by 45 min. Alkaline phosphatase treatment revealed this modification was caused by a change in occludin phosphorylation. In addition, the quantity of extracted occludin increased 2-fold in the same time frame. The phosphorylation and increased extraction of occludin was recapitulated in retinal endothelial cells in culture after VEGF stimulation. The data presented herein are the first demonstration of a change in the phosphorylation of this transmembrane protein under conditions of increased endothelial permeability. In addition, intra-ocular injection of VEGF also caused tyrosine phosphorylation of ZO-1 as early as 15 min and increased phosphorylation 4-fold after 90 min. In conclusion, VEGF rapidly increases occludin phosphorylation as well as the tyrosine phosphorylation of ZO-1. Phosphorylation of occludin and ZO-1 likely contribute to regulated endothelial paracellular permeability.     

The distribution of renin in the different segments of the renal arterial tree

Summary The intrarenal distribution of renin in the mouse kidney was evaluated in a semiquantitative immunocytochemical study using an antiserum against pure mouse renin and the PAP technique. The bulk of renin positive cells was found in the media of the afferent arteriole. When examining the geometry of renin distribution about 35% of the afferent vessels were seen to be renin positive only over a distance of 20 m, about 60% over a distance of 30 m. In the remaining afferent arterioles, renin was also found upstream over distances up to 100 or even 200 m. These results are discussed with regard to the stimuli for renin secretion, especially the macula densa signal. — At the vascular pole of the glomerulus, virtually 100% of the afferent, and 20%–40% of the efferent arterioles were found to be renin positive at an antiserum dilution of 1:1,000. As some efferent vessels — especially those of the juxtamedullar region — show scattered activity occasionally over a distance of more than 100 m, it is suggested that the figure of 20%–40% should be taken as a minimal count for renin positive efferent arterioles. — To compare the renin content of superficial and juxtamedullary, afferent and efferent arterioles in normal salt and salt depleted mice, the fraction of positive renin reactions close to the vascular pole was determined at antiserum concentrations of 10^–3, 10^–4, 2×10^–4 and 10^–5. By this semiquantitative immunocytochemical method the afferent arterioles of superficial glomeruli could be shown to contain significantly higher renin concentrations than those of juxtamedullar glomeruli. This result was in agreement with biochemical renin estimations in mouse kidney slices taken from cortical and juxtamedullar sites. Sodium deprivation was followed by only a slight elevation of the fraction of positive superficial afferent arterioles (confirmed by the biochemical data). In contrast, sodium deprivation induced a highly significant increase of the number of positive superficial efferent vessels. This result is discussed with regard to (controversial) reports on a preferential efferent vasoconstrictor tone sustained by angiotensin II especially under the condition of sodium depletion. Juxtamedullar vasa afferentia and efferentia did not respond significantly to sodium restriction. —The Goormaghtigh cell field was found to be renin negative in superficial as well as in juxtamedullar glomeruli both in normal salt and salt deprived mice. Inspecting nearly 5,000 glomeruli, only 5 clearly renin positive mesangial cells were seen close to the glomerular stalk. In contrast, renin positive media cells could not seldom be seen in interlobular arteries and at the point of their branching into afferent arterioles.A first account of these results was given at the Rottach-Egern Satellite Symposium of the VIIth International Congress of Nephrology: The juxtaglomerular apparatus and the tubuloglomerular feedback mechanism — morphology, biochemistry and function, June 3 to 5, 1981These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

Contribution of cardiopulmonary baroreceptors to the control of the kidney.

The role of cardiopulmonary receptors in the control of renal sympathetic nerve activity and of renin release is reviewed. The evidence indicates that cardiopulmonary receptors with vagal afferents exert a tonic inhibition on both renal nerve activity and on renin release. The magnitude of this inhibition appears directly related to changes in blood volume. Atrial as well as ventricular receptors can influence the secretion of renin. Cardiopulmonary receptors with vagal afferents may also reflexly modulate renal prostaglandin secretion. There is preliminary evidence to suggest that cardiopulmonary receptors with sympathetic afferents can influence renal nerve activity. The limitations of previous studies are outlined and a direction for future studies is suggested. It is concluded that alterations in cardiopulmonary vagal afferent input and the resulting changes in renal nerve activity and in renin release are appropriate for the maintenance of blood volume homeostasis.     

Plasma renin activity, aldosterone and cortisol secretions after acute hemorrhage and supine to vertical postural change in dogs

Acute hemorrhage and horizontal to vertical postural change are accompanied by decrease in blood volume of cardiovascular central reflexogenic areas (CRA) and by central hypoxia, followed by pressor responses. In these both circumstances important reflexogenic and humoral pressor reactions occured, as cathecolamine, renin and aldosterone hypersecretions. Aldosterone hypersecretion is considered as produced by angiotensin II, by a complex renin-angiotensin(RA)-aldosterone system. The main purpose of this work was to clarify the presence of this RA-aldosterone system after acute hemorrhage and in head-up postural change. In this aim we studied on dogs renin, aldosterone and cortisol responses. We analysed in these two circumstances the correlation of plasma renin activity(PRA) and aldosterone plasma concentration(p.c.) in intact and bilaterally nephrectomised(BN) dogs. We also studied correlations between aldosterone and cortisol p.c., having in view that both are stimulated by ACTH, searching in this way another modality for aldosterone secretion.     

Endothelial cell signaling during conducted vasomotor responses

ACh and KCl stimulate vasomotor responses that spread rapidly and bidirectionally along arteriole walls, most likely via spread of electric current or Ca2+ through gap junctions. We examined these possibilities with isolated, cannulated, and perfused hamster cheek pouch arterioles (50- to 80-microm resting diameter). After intraluminal loading of 2 microM fluo 3 to measure Ca2+ or 1 microM di-8-ANEPPS to measure membrane potential, photometric techniques were used to selectively measure changes in intracellular Ca2+ concentration ([Ca2+]i) or membrane potential in endothelial cells. Activation of the endothelium by micropipette application of ACh (10-4 M, 1.0-s pulse) to a short segment of arteriole (100-200 microm) increased endothelial cell [Ca2+]i and caused hyperpolarization at the site of stimulation. This response was followed rapidly by vasodilation of the entire arteriole ( approximately 2-mm length). Change in membrane potential always preceded dilation, both at the site of stimulation and at distant sites along the arteriole. In contrast, an increase in endothelial cell [Ca2+]i was observed only at the application site. Micropipette application of KCl, which can depolarize both smooth muscle and endothelial cells (250 mM, 2.5-s pulse), also caused a rapid, spreading response consisting of depolarization followed by vasoconstriction. With KCl stimulation, in addition to changes in membrane potential, increases in endothelial cell [Ca2+]i were observed at distant sites not directly exposed to KCl. The rapid longitudinal spread of both hyperpolarizing and depolarizing responses support electrical coupling as the mode of signal transmission along the arteriolar length. In addition, the relatively short distance between heterologous cell types enables the superimposed radial Ca2+ signaling between smooth muscle and endothelial cells to modulate vasomotor responses.