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     

Coexistence of renin and angiotensin II in epitheloid cell secretory granules of rat kidney

Summary The distribution of renin and angiotensin II (ANG II) in juxtaglomerular epitheloid cells of control and adrenalectomized rats was studied, using specific antisera and the protein A-gold technique in Lowicryl- and glycol methacrylate-embedded tissue.The matrix of virtually all mature secretory granules of epitheloid cells contains not only renin, but also ANG II. On adrenalectomy, the concentration of both renin and ANG II in the granule internum increases markedly, as indicated by the density of the immunolabel.Given the coexistence of renin and ANG II in the granule matrix, it is quite probable that, with each secretory event, a certain amount of ANG II is released together with renin. Further experiments will have to show if this amount of ANG II cosecreted with renin is sufficient to elicit immediate local intrarenal actions.ANG I, as well as angiotensinogen and converting enzyme, were not found in epitheloid cells. It is therefore inferred that ANG II is not generated intracellularly, but within the extracellular space and subsequently taken up by pinocytosis and incorporated into the secretory granules of epitheloid cells.These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

Renin-angiotensin system affects endothelial morphology and permeability of renal afferent arteriole

The afferent arteriole (AA) is an important regulatory site of renal function and blood pressure. We have demonstrated endothelial fenestration and high permeability in the vicinity of renin granulated epithelioid cells in the juxtaglomerular portion of the afferent arteriole in different mammals. The permeability of fenestrated endothelium of afferent arteriole may be important in connection to various physiologic and pathophysiologic processes. We have assumed that the permeable fenestration may serve as a communication channel between the intravascular circulation and a pathway for renin secretion. Utilising the multiphoton image technique we were able to visualise the endothelial fenestration and renin granules of the in vitro microperfused AA and in vivo AA. We demonstrated that ferritin-positive, i.e., permeable portion of the afferent arteriole, under control conditions is on average 45 microm, which is about one-third to half of the total length of the afferent arteriole. The length of this portion is not constant and can change by physiologic and pharmacologic manipulation of renin formation. The permeability of the afferent arteriole is not changing only parallel with the pharmacologically stimulated renin secretion as already demonstrated in adult rats, but also with the change of renin appearance in afferent arteriole within the very first few days of life after birth. Independently from the age there is a significant correlation between the renin-positive and permeable portion of the AA. Further studies are necessary to clarify the physiological significance of afferent arteriolar permeability and its changes in the postnatal development of the kidney, as well as in correlation with activity of renin- angiotensin system.     

Connexin45 is expressed in the juxtaglomerular apparatus and is involved in the regulation of renin secretion and blood pressure

Connexin (Cx) proteins are known to play a role in cell-to-cell communication via intercellular gap junction channels or transiently open hemichannels. Previous studies have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the vascular connexin isoform Cx45 has not yet been studied in this region. The present work aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional role for Cx45 in the kidney using conditions where Cx45 expression or function was altered. Using mice that express lacZ coding DNA under the control of the Cx45 promoter, we observed beta-galactosidase staining in cortical vasculature and glomeruli, with specific localization to the JGA region. Renal vascular localization of Cx45 was further confirmed with the use of conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice, which express enhanced green fluorescence protein (EGFP) instead of Cx45 only in cells that, during development, expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriole smooth muscle cells, in the renin-producing juxtaglomerular cells, and in the extra- and intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin expression and activity, as well as higher systemic blood pressure. The propagation of mechanically induced calcium waves was slower in cultured vascular smooth muscle cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice and in control VSMC treated with a Cx45 gap mimetic peptide that inhibits Cx45 gap junctional communication. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye Lucifer yellow, and calcium wave propagation was not altered by addition of the ATP receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the propagation of JGA vascular signals and in the regulation of renin release and blood pressure.     

The juxtaglomerular apparatus of rats with hereditary hypothalamic diabetes insipidus

Summary The juxtaglomerular apparatus (JGA) of rats with hereditary hypothalamic diabetes insipidus (DI) was studied. Plasma concentration of renin and angiotensin II, as well as serum sodium concentration and serum osmolality of DI rats are elevated. The morphological examination reveals no characteristic alteration of the epitheloid cells. The results show that the epitheloid cells are sufficiently adapted for the higher release of renin.Supported by the Deutsche Forschungsgemeinschaft SFB 90 Heidelberg. The expert technical assistance of Mrs. Marlis Kopp is gratefully acknowledged     

Ultracytochemistry of Aminopeptidase A (angiotensinase A) in the kidney glomerulus and juxtaglomerular apparatus

Summary Ultracytochemical studies of the Aminopeptidase A (APA; angiotensinase A; E.C. 3.4.11.7) were performed on perfusion fixed rat and mouse kidneys (low-concentration aldehydes) using simultaneous azo coupling with -l-Glu-MNA as substrate and HPR as well as HNF as coupling agents. The studies of glomeruli and juxtaglomerular apparatus (JGA) show that APA is localized mainly at the cell membranes of podocytes and endothelial cells (rat and mouse) and of epitheloid cells (mouse) and Goormaghtigh's cells (rat). Increased APA activities are found in the region of cell contacts of epitheloid cells (mouse) and Goormaghtigh's cells (rat). In the epitheloid cells of mice, reaction product is also observed intracellularly in lysosomal structures. Concerning the functional significance of APA in the glomerulus and JGA, it would appear that this enzyme modifies or regulates angiotensin effects in the glomerulus and JGA through angiotensin degradation.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

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     

Increased expression of cyclooxygenase 2 contributes to aberrant renin production in connexin 40-deficient kidneys

We previously found that deletion of connexin 40 (Cx40) causes a misdirection of renin-expressing cells from the media layer of afferent arterioles to the perivascular tissue, extraglomerular mesangium, and periglomerular and peritubular interstitium. The mechanisms underlying this aberrant renin expression are unknown. Here, we questioned the relevance of cyclooxygenase-2 (COX-2) activity for aberrant renin expression in Cx40-deficient kidneys. We found that COX-2 mRNA levels were increased three-fold in the renal cortex of Cx40-deficient kidneys relative to wild-type (wt) kidneys. In wt kidneys, COX-2 immunoreactivity was minimally detected in the juxtaglomerular region, but renin expression was frequently associated with COX-2 immunoreactivity in Cx40-deficient kidneys. Treatment with COX-2 inhibitors for 1 wk lowered renin mRNA levels in wt kidneys by about 40%. In Cx40-deficient kidneys, basal renin mRNA levels were increased two-fold relative to wt kidneys, and these elevated mRNA levels were reduced to levels of untreated wt mice by COX-2 inhibitors. In parallel, renin immunoreactive areas were clearly reduced by COX-2 inhibitors such that renin expression vanished and decreased significantly in the periglomerular and peritubular extensions. Notably, COX-2 inhibitor treatment lowered plasma renin concentration (PRC) in wt kidneys by about 40% but did not affect the highly elevated PRC levels in Cx40-deficient mice. These findings suggest that aberrant renin-producing cells in Cx40-deficient kidneys express significant amounts of COX-2, which contribute to renin expression in these cells, in particular, those in the periglomerular and peritubular position. Apparently, these disseminated cells do not contribute to the enhanced renin secretion rates of Cx40-deficient kidneys.     

Vascular and tubular renin in the kidneys of mice

Summary Monospecific antisera and the immunocytochemical PAP-method have been used to localize renin in the kidneys of mice. With this procedure, reaction product was not only observed in the epitheloid cells of kidney vessels but also in kidney tubules: in the apical portion of proximal tubule cells and in some cells of the connecting and the cortical collecting tubule. To answer the question, whether the occurrence of renin in kidney tubule cells is the consequence of tubular synthesis or that of glomerular filtration of plasma renin followed by its uptake from the tubular lumen, tracer experiments with radioiodinated renin and with horseradish peroxidase were undertaken. The results of these studies as well as other arguments suggest reabsorptive pinocytosis of the filtered hormone as the source of tubular renin.These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

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.     

The morphological basis of fluid balance in the interstitium of the juxtaglomerular apparatus

Summary The morphological basis of fluid balance in the interstitium of the juxtaglomerular apparatus (JGA) was reevaluated in rats, mice and Tupaia. Three ultrastructural features in the region of the vascular pole of the renal corpuscle are described that may be important for the fluid balance in this region: (1) podocyte foot processes in the parietal layer of Bowman's capsule, (2) endothelial fenestrations in the wall of the incoming afferent arteriole, both facing Goormaghtigh and epithelioid cells, and (3) the mesangial type lining of the glomerular stalk. With respect to the relevant pressure gradients, this morphology may provide the basis of bulk-fluid flow directed to the interstitium of the JGA including the Goormaghtigh cell field. Thus, the fluid balance in the lacis area and, consequently, the tubulo-glomerular feedback mechanism, probably does not solely depend upon the reabsorptive transport of the macula densa. Similar considerations may be valid for the humoral control of renin secretion from juxtaglomerular epithelioid cells.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Species variability in the expression of met- and leu-enkephalin-like immunoreactivity in mammalian Merkel cell dense-core granules

Summary Immunogold staining failed to show met-enkephalin immunoreactivity in the Merkel cell dense-core granules of rats when examined by electron microscopy, but showed gold particle staining in the Merkel cell dense-core granules of mice and nude mice. Merkel cells of hamster, guinea pig, rabbit, cat and dog were also examined using a similar method, and different antisera dilutions. Immunogold particles were consistently found in the dense-core granules of mice and nude mice at all antisera dilutions, but not in the other species, except in the dog, where a very low labelling response was encountered. Merkel cells from skin touch domes or sinus hair follicles, did not exhibit any difference in peptide expression as far as met-enkephalin immunoreactivity was concerned. In addition, all species studied, including mice and nude mice, did not show leu-enkephalin immunoreactivity in their Merkel cell dense-core granules. It is concluded that species variability in peptide expression occurs in the Merkel cell dense-core granules, and may be closely related to the different methodologies used.     

Immunoreactive renin and angiotensin II in the afferent glomerular arterioles of rats with hypertension due to unilateral renal artery constriction

Summary The immunoreactivity for renin and angiotensin II (ANG II) in the ischaemic and non-ischaemic kidney of rats with renovascular hypertension was compared with that of the kidneys of sham operated controls. In addition, the renin concentration of these kidneys and the plasma level of ANG II were determined in hypertensive and control animals. In parallel with the renin concentration of kidney cortex, the immunoreactivity, i.e. the JG-index for renin of the afferent arterioles from the ischaemic kidney was slighly increased, that from the nonischaemic kidney drastically decreased as compared to control kidneys. Similarly, the JG-index for ANG II was increased in the ischaemic and decreased in the non-ischaemic kidney although the plasma level of ANG II was elevated in the animals with renovascular hypertension. This difference in the immunocytochemically detectable ANG II and especially the decrease of ANG II in the non-ischaemic kidney in spite of elevated plasma ANG II levels is interpreted to result from similar differences in the local (extravascular) formation of ANG II by the intrarenal renin-angiotensin system.These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

Renin immunohistochemistry in the mesonephros and metanephros of the pig embryo

Summary The occurrence and distribution of renin was investigated in meso- and metanephric kidneys of pig embryos in various gestational stages. The immunohistochemical peroxidase-antiperoxidase-method (PAP) was used on paraffin sections after application of an antiserum against mouse renin which cross reacts with pig renin. Renin immunoreactivity was already found in the mesonephros of 21 day pig embryos (crown-rump(CR)-length 12 mm) with the strongest reaction in the media of the juxtaglomerular afferent arteriole. Efferent vessels, mesonephric arteries, and the aortic wall also contained scattered renin-positive cells. In the definitive kidney, renin was not detected prior to the 25 mm CR-length-stage. In 45 mm embryos, immunocytochemical staining was observed not only in the media of kidney arteries and arterioles, but also in proximal tubules after pinocytic absorption of filtered renin. TEM-studies revealed that the media of both the mesonephric and the developing metanephric arteries and arterioles contains epithelioid cells whose ultrastructure is very similar to that of renin-producing cells in the adult organ. The observed distribution of renin-producing cells along the entire renal arterial tree points to the possibility that the major function of the renin-angiotensin system in the fetal animal is to participate in the stabilization of renal perfusion pressure.     

Measurement and localization of angiotensin-like immunoreactivity in juxtaglomerular cells of the rat

The existence and distribution of angiotensin I (A I) and angiotensin II (A II) in rat kidney were examined in immunocytochemical studies using the peroxidase-antiperoxidase technique and in biochemical studies using rat kidney homogenates extracted with acid-ethanol and purified by Sephadex G-25 gel chromatography. Immunopositive A II-like staining was observed in the juxtaglomerular cells of the afferent arteriole, but no histochemical evidence for A I was found. On the other hand, renal homogenates were found to contain both A I and A II immunoreactivities which coeluted on gel chromatography with synthetic A I and A II. These results indicate that A I as well as A II immunoreactivities are present in the kidney and that A II immunoreactivity can be localized to the juxtaglomerular cells. The origin of the immunoreactive A II in the juxtaglomerular cells remains to be determined.     

Renin in the human kidney

Summary We have localized the enzyme renin (EC 3.4.99.19) in the normal adult human kidney by immunohistology. Serial paraffin sections of kidneys were incubated with renin antisera and then processed by the peroxidase-antiperoxidase method. Renin immunoreactivity was observed in the juxta-glomerular epithelioid granular cells (JEG-cells) in the wall of the afferent and rarely of the efferent vessel of the glomerulus. JEG-cells have long cytoplasmic processes penetrating between adjacent cells. This suggests a possible paracrine release of renin. Staining of various segments of the tubular system was shown to be artifactual. The kidney proteins recognized by our anti-human renin antisera had similar characteristics to renin when determined by a combination of gel-electrophoretic and immunologic techniques. Renin immunostaining in the juxtaglomerular apparatus of the human kidney is discrete and reflects the low amount of extractable renin.Dedicated to Prof. G. Töndury on the occasion of his 75th birthday     

Ultrastructural changes associated with renin secretion from the juxtaglomerular apparatus of mice

Summary Thin sections and freeze-fracture replicas were used to investigate the ultrastructural changes associated with renin secretion from the juxtaglomerular part of the afferent arteriole of male mice. Adrenalectomized animals in which renin secretion was stimulated by furosemide application and bleeding were also studied. Exocytosis of mature electron-dense granules was found in all experimental groups. Before extrusion, the region of granule facing the cell membrane changed, with vesicular and/or stacked membrane-like profiles and a small local protrusion of the granule membrane appearance of. Concomitantly, punctuate sites of fusion between the cell and granule membranes were observed. Later, unaltered amorphous, and altered membrane-like granule content was released from omega-shaped cavities into the extracellular space. In stimulated animals the alteration and extrusion of several closely apposed granules was reminiscent of compound exocytosis. Coated pits were frequently seen, suggesting specific retrieval of the former granule membrane. The collapsing silhouette of a depleted granule very rarely took the form of a saccule whose narrow membrane-bounded neck was continuous with the extracellular space.Observed were two additional events by which active and inactive renin may be released. Small electron-lucent vacuoles of undetermined origin fused with the cell membrane and, in stimulated kidneys, some epithelioid cell processes disintegrated. However, the interpretation of the related ultrastructural phenomena was uncertain.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Immunohistochemical study of a large molecular fragment of thyroglobulin in parafollicular cells

Thyroglobulin-like immunoreactivity of the parafollicular cells was studied by an immunoperoxidase bridge technique using antisera against dog thyroglobulin fragments. 1. The dog parafollicular cells were specifically stained by anti-peak I (27S and larger components fraction) antiserum absorbed with peak II (19S fraction). By this method, they were easily distinguishable from the non-reactive follicular cells and colloid droplets. More sensitive staining of the parafollicular cells was possible with anti-peak I' (larger components fraction) antiserum. The staining reactions indicated that the antigenic material responsible for immunoreactivity of the parafollicular cells was due to larger molecular components of thyroglobulin corresponding to 32S, 37S or greater than 37S, and was not due to either the 19S thyroglobulin or to the 27S iodoprotein. 2. A conspicuous decrease of the immunoreactive material in the parafollicular cells occurred in the dog after both chronically induced hypercalcemia and antithyroid drug treatment. This coincided with movement of secretory granules containing calcitonin as shown by staining with silver impregnation, HCl-basic dye, and lead-hematoxylin. 3. The antisera against larger molecular components of dog thyroglobulin showed a high degree of cross-reactivity to the parafollicular cells of most of the mammalian species investigated; rats, rabbits, hamsters, mice, cats, lions, goats, cows, and human.     

The fate of prorenin during granulopoiesis in epithelioid cells. Immunocytochemical experiments with antisera against renin and different portions of the renin prosegment

Comparative immunocytochemical experiments with antisera directed against renin and three synthetical peptides (Pro 1, Pro 2A and Pro 3) covering almost the entire span of human renin prosegment were performed on human kidney tissue. With anti-Pro 1, i.e. the antiserum which recognizes the NH2 terminus of human prorenin, no clear immunolabeling of juxtaglomerular epithelioid cell secretory granules could be obtained. It is therefore concluded that the corresponding portion of human prorenin may be cleaved off in the Golgi complex. After application of anti-Pro 3, the antiserum which recognizes the COOH terminus of the prosegment, only the juvenile secretory granules of epithelioid cells were consistently labeled, whereas, in contrast, some of the intermediate and most of the mature secretory granules were anti-Pro 3-negative. As the immunoreactivity of mature renin increased remarkably from protogranules to mature secretory granules, it is suggested that the cleavage of the COOH terminus of the prosegment, i.e. the activation of renin, takes place in juvenile and intermediate granules during condensation of the enzyme. The immunoreactivity of Pro 2A, corresponding to the middle portion of the prosegment, disappeared in a somewhat earlier stage of granulopoiesis than that of Pro 3. It is therefore concluded that the corresponding segmental cleavage, the result of which is a truncated version of intact prorenin, occurs in the protogranules of epithelioid cells.(ABSTRACT TRUNCATED AT 250 WORDS)