Characterization of the macula densa stimulus for renin secretion.

These studies utilize the isolated perfused rabbit juxtaglomerular apparatus (JGA) to study the macula densa signal for renin secretion in the absence of the confounding influences of intravascular pressure and renal nerve activity. In the first experimental series, JGAs were perfused alternately with high- and low-NaCl solutions to determine the reversibility of the renin response to changes in NaCl concentration. Compared with high-NaCl controls, perfusion with a low-NaCl solution resulted in a fivefold increase in renin secretion rate (RSR) [2.1-10.0 nano-Goldblatt hog units (nGU)/min], and this response was largely reversible. When the solutions were presented in the reverse order, a similar inhibition by high NaCl was observed. In the second series, JGAs were perfused with high-, medium-, and low-NaCl solutions to determine the sensitive range of the renin response to NaCl concentration changes. The full renin response (3.2-16.6 nGU/min), similar in magnitude to that seen in series 1, was found to occur between 80 and 24 mM for Na+ and 61 and 7 mM for Cl-. In the third series, the NaCl concentration and flow rate of the perfusate were altered independently to separate the effects of flow rate, NaCl delivery, and NaCl concentration on RSR. Although a decrease in perfusate flow rate slightly increased RSR (3.4-8.1 nGU/min), a comparable decrease in NaCl concentration resulted in a much higher RSR (26.3 nGU/min). We conclude that in this preparation 1) RSR responds equally to both increases and decreases in macula densa NaCl concentration, and these changes are rapid and largely reversible, 2) the full renin response occurs within the concentration range normally occurring at the macula densa, i.e., below 80 mM Na+ and 61 mM Cl-, and 3) RSR responds with a larger change to alterations in NaCl concentration than in NaCl delivery or fluid flow rate.

Expression of renin and its mRNA in the adult rat kidney with chronic ureteral obstruction.

Angiotensin II has been implicated in mediating renal vasoconstriction resulting from chronic unilateral ureteral obstruction (UUO) in both mature and developing animals. We have previously shown that chronic neonatal UUO results in increased distribution of renin and its mRNA in the obstructed kidney, as well as of immunoreactive renin in the intact opposite kidney. The present study was designed to evaluate the effects of 24 hours versus 4 weeks of UUO on the distribution of renin mRNA and its protein in the adult rat kidney. Renin was detected by immunocytochemistry using a polyclonal anti-rat renin antibody. Renin mRNA was localized by in situ hybridization to an oligonucleotide complementary to renin mRNA. UUO of 24 hours' or 4 weeks' duration did not alter the distribution of renin and its mRNA in the obstructed kidneys as compared with sham-operated kidneys, although kidneys obstructed for 4 weeks had a significant increase in the percent of renin-containing juxtaglomerular apparatuses (JCA) when compared with the intact opposite kidneys (P less than 0.05). Compensatory hypertrophy was not present in the intact opposite kidneys after 24 hours of UUO and distribution of renin gene expression was not altered at that time. However, 4 weeks following contralateral UUO, the intact kidneys were hypertrophied and showed a decrease in renin gene expression relative to the obstructed and sham-operated kidneys. We conclude that unlike UUO during early development, chronic UUO in the mature animal does not activate renin gene expression nor alter renin distribution in the obstructed kidneys. Renin gene expression is suppressed in the hypertrophied kidney with prolonged contralateral UUO.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of decay accelerating factor in normal and diseased kidneys.

Decay accelerating factor (DAF) is a cell membrane associated glycoprotein that inhibits C3 activation. In the present study we evaluated the presence of DAF in normal (N = 15) and diseased human kidneys (N = 76). Sections of frozen tissue were stained for DAF by immunoperoxidase, utilizing three mouse monoclonal anti-DAF anti-bodies. In normal kidneys, DAF was localized in the glomerular vascular pole, apparently in the juxtaglomerular apparatus (JGA). All other structures were negative for DAF. By contrast, in diseased kidneys, two types of abnormalities were detected. First, JGA-DAF was significantly decreased and this abnormality correlated with the pathologic diagnosis and with the presence of C3, IgM and/or fibrinogen in the glomeruli. Second, DAF was present in the glomerular mesangium (67%), renal interstitium (68%) and/or blood vessels (38%). The presence of DAF in the mesangium and interstitium of the kidney correlated with each other and correlated with C1q and C3 deposition in the glomerulus. Finally, vascular DAF was significantly more common in patients with electron dense deposits in the glomeruli. In summary, DAF is present in the normal kidney and is located exclusively in the glomerular vascular pole. In diseased kidneys, DAF tends to be lost from the JGA but is often present in glomerular mesangium, interstitium and blood vessels. This pattern is specially prominent in patients demonstrating complement deposition in the glomerulus. We speculate that kidney DAF may play a role in protecting the kidney against the products of complement activation.

Macula densa cells sense luminal NaCl concentration via furosemide sensitive Na+2Cl-K+ cotransport.

The macula densa cells of the juxtaglomerular apparatus probably serve as the sensor cells for the signal which leads to the appropriate tubuloglomerular feedback response. The present study reports basolateral membrane voltage (PDbl) measurements in macula densa cells. We isolated and perfused in vitro thick ascending limb segments with the glomerulus, and therefore the macula densa cells, and the early distal tubule still attached. Macula densa cells were impaled with microelectrodes under visual control. PDbl was recorded in order to examine how these cells sense changes in luminal NaCl concentrations. The addition of furosemide, a specific inhibitor of the Na+2Cl-K+ cotransporter in the thick ascending limb, to the lumen of the perfused thick ascending limb hyperpolarized PDbl from -55 +/- 5 mV to -79 +/- 4 mV (n = 7). Reduction of NaCl in the lumen perfusate from 150 mmol/l to 30 mmol/l also hyperpolarized PDbl from -48 +/- 3 mV to -66 +/- 5 mV (n = 4). A Cl- concentration step in the bath from 150 mmol/l to 30 mmol/l resulted in a 24 +/- 4 mV (n = 4) depolarization of PDbl. This depolarization of PDbl was absent when furosemide was present during the Cl- concentration step. These data suggest that the macula densa cells sense changes in luminal NaCl concentration via coupled uptake of Na+ and Cl-.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of type IV collagen fibronectin and laminin in the juxtaglomerular apparatus of the rat kidney.

The juxtaglomerular apparatus (JGA) is a complex structure containing several components: the vessels, the extraglomerular mesangium and the distal tubule. These structures include cellular elements and an extracellular matrix (ECM). Collagenous (type IV collagen) and noncollagenous components of the basement membranes were studied. The localization of type IV collagen and of two extracellular glycoproteins (laminin and fibronectin) was investigated using immunofluorescent and immunoperoxidase labelled antibodies. Type IV collagen and laminin have the same localization on the JGA basement membranes. On the other hand, fibronectin is limited to the entrance of the glomerular stalk. On electron microscopy, type IV collagen is found in the basement membrane while fibronectin is restricted to certain areas of the extracellular matrix. These findings confirm data concerning the distribution of these three components in basement membranes and allow a better understanding of the histoarchitecture of the juxtaglomerular apparatus.

Immunohistochemical localization of renin in end-stage kidneys.

Hypertension in chronic renal failure is usually due to excessive accumulation of salt and water. In some cases, sodium and volume depletion by dialysis fail to reduce the high BP, and plasma renin activity tends to be higher. We performed a semiquantitative analysis of the immunohistochemical distribution of renin in the kidneys of ten patients with end-stage renal disease and hypertension using a specific antihuman renin antibody and a peroxidase-antiperoxidase technique on paraffin sections of nephrectomy and/or autopsy specimens. In five cases with severe, dialysis-resistant hypertension, the degree of immunoreactivity was most striking, exceeding that found in renovascular hypertension and present in arterioles at a distance from the glomeruli. Three cases of advanced diabetic glomerulosclerosis consistently showed minimal immunoreactivity. We conclude that renin often can be detected immunologically in the kidney of patients with chronic renal failure and hypertension, but its pathophysiological role will require further study.

Anatomical relationship between kallikrein-containing tubules and the juxtaglomerular apparatus in the human kidney.

Current evidence suggests a functional and biochemical link between the renin and the kallikrein systems. The purpose of this work was to study the localization of kallikrein along the human nephron to elucidate whether there exists an anatomical base for such interrelation. Serial sections of human kidney tissue were stained by immunocytochemical methods with antisera against kallikrein. Kallikrein immunostaining was observed exclusively in segments of the distal nephron lying in the cortical labyrinths and forming arcades in its distal portion. Consistently the tubules containing kallikrein established a close anatomical relationship with the afferent arteriole of the juxtaglomerular apparatus providing an anatomical base for an interaction between the renin and kallikrein systems in the human kidney.

Isolation of renin-rich rat kidney cells.

Enzymatic dispersion and density gradient (Percoll) sedimentation were used to isolate a population of renin-containing, granule-laden cells (density 1.067 g/ml) from rat kidney cortex. Using immunohistochemistry (light microscopy) and electron microscopy, we defined the presence and ability of these cells to store renin protein(s). A 1000 base pair rat renin complementary DNA was used to show that these cells express the renin gene. The reverse hemolytic plaque assay defined the functional properties of the renin-containing cell. The data are consistent with the postulated inverse relationship between calcium concentration and release of renin. Thus, we have isolated a population of functional rat kidney cells that synthesize, store, and release renin.

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)

Discrepancy between aldosterone production and renin-angiotensin system activity in Brattleboro rats.

It was demonstrated before that in addition to their typical changes in water-sodium-potassium balance Brattleboro rats, homozygous for hypothalamic diabetes insipidus (DI), revealed a discrepancy between aldosterone level and plasma renin activity (PRA). In the present study PRA was significantly increased (79%), and concomitantly juxtaglomerular (JG) index was increased, reflecting an increased secretory activity of renin-producing JG cells. Plasma concentration of aldosterone was significantly lower (-36%) in DI rats than in their Long Evans (LE) controls. Adrenal blood flow rates were not significantly different in both groups of rats but aldosterone concentrations in the adrenal venous effluents were significantly lower (-66%) in DI rats than in LE rats, suggesting that in vivo production rate of aldosterone was reduced in DI rats. This assumption was confirmed by morphometric data of zona glomerulosa. Our results demonstrated a significant reduction (50%) of angiotensin II receptors in the adrenal glands of DI rats, referring to the number of binding sites and to Kd. This finding threw light on the dissociation between a decreased aldosterone production and stimulated renin-angiotensin system in DI rats.

Cathepsin D coexists with renin in the secretory granules of juxtaglomerular epithelioid cells.

Mature juxtaglomerular epithelioid cell secretory granules of the rat exhibit both renin- and cathepsin D-like immunoreactivity. On the basis of the coexistence with renin at a pH which, according to previous experiments, is probably in the range of that in lysosomes, cathepsin D is suggested to be involved in the regulation of the granular renin stores available for secretion.

Immunoelectron microscopic localization of renin in the juxtaglomerular cells of the amphibian Bufo bufo.

The ultrastructural localization of renin in the juxtaglomerular apparatus of the kidney of the toad Bufo bufo has been examined using an immunogold staining method for electron microscopic immunocytochemistry and an antiserum to renin isolated from the submandibular gland of the mouse. Renin immunoreactivity was confined to lamellated granules in the cytoplasm of epitheloid or juxtaglomerular cells in the glomerular afferent arterioles and also in the media cells of larger arteries. Mouse kidney tissue, examined for purposes of comparison, showed immunolabeling limited to the granules of the juxtaglomerular cells. The presence of renin or a renin-like substance in the juxtaglomerular granules of the toad kidney is discussed in relation to the lysosomal nature of these granules. A model is presented linking the lysosomal function of the juxtaglomerular granules and the release of renin mediated by beta-adrenergic receptors present on the surface of the juxtaglomerular cells.

Characterization of beta-adrenoceptor subtypes in rat kidney with new highly selective beta 1 blockers and their role in renin release.

Highly selective beta-adrenoceptor blocking agents with a beta 1: beta 2-selectivity ratio of 0.015 to 3400 were used to characterize the beta-adrenoceptors present in rat kidney and to identify those mediating renin release. The results obtained with ICYP binding to kidney membranes revealed the presence of both beta 1- and beta 2-adrenoceptors in a ratio of 1:1. The pKD beta 1- and pKD beta 2-values of selective beta-antagonists obtained in rat kidney membranes correlated well with those found in guinea pig left ventricle (beta 1) and lung (beta 2), indicating that kidney receptor subtypes are pharmacologically identical with those in the ventricle and lung, respectively. In the isolated perfused rat kidney, the apparent pA2 values of beta 1-selective blockers for inhibition of isoprenaline-stimulated renin release correlated well with pKD beta 1, but not with pKD beta 2 values. These results clearly show that the beta 1-adrenoceptor subtype mediates renin release in the rat kidney.

Adrenergic effects on renal secretion of epidermal growth factor in the rat.

Urinary epidermal growth factor (EGF) has been demonstrated recently to originate from the kidneys. The present study was undertaken to investigate the adrenergic and cholinergic influence on secretion of renal EGF. beta-Adrenergic agonists increased the level of urinary EGF, while propranolol, a beta-adrenergic blocking agent, decreased basal and beta-adrenergic stimulated total output of urinary EGF. Acetylcholine and the anticholinergic agent atropine had no effect on the output of EGF in urine. Also chemical sympathectomy induced by 6-hydroxydopamine reduced the urinary output of EGF. None of the experimental groups had a median serum concentration above the detection limit of the assay. The present study shows that secretion of renal EGF is under the influence of the sympathetic nervous system and release of EGF is stimulated by activation of beta-adrenergic receptors in the kidneys.

Abnormalities of renin-containing cells in human glomerular and vascular renal diseases.

The distribution of renin was investigated by immunofluorescence in human kidney biopsy specimens (27 patients with lipoid nephrosis, 39 with Berger disease, 17 with membranous glomerulonephritis, 5 with thrombotic microangiopathy, and 7 with malignant nephroangiosclerosis). A semiquantitative assessment was carried out. Two ratios were found significatively increased in the study groups as compared with the control group: JGA + and JGA ++ which expressed, respectively, the number of fluorescent JGA in relation to the number of glomerular sections and the number of fluorescent JGA with more than six renin-containing cells (RCC) in relation to the number of immunoreactive JGA. Highest values were observed in patients with thrombotic microangiopathy and malignant nephroangiosclerosis (P less than 0.001). The above immunomorphological parameters were correlated with clinical and laboratory data. A positive dependency was found between JGA + and JGA ++ ratios and a low sodium diet, diuretic therapy and serum creatinine. A negative dependency was seen in the albumin and hemoglobin serum levels. No correlation was found with blood pressure values. These observations suggested that decreased plasma volume and impaired renal function could be factors leading to an increased renin production in the kidney.

Angiotensin II immunoreactivity coexisting with renin in the human juxtaglomerular epithelioid cells.

The various components of the renin-angiotensin system in the human kidney have been detected by immunohistochemical techniques using specific antisera. Renin and AII immunoreactivities coexist in the JEG cells of the adult human kidney. I propose that AII is synthesized intracellular in the JEG cells of the human kidney and is secreted locally in the juxtaglomerular region, affecting the width of the glomerular arterioles and the contraction state of the glomerular mesangium.

Immunohistochemical characterization of renin-containing cells in the human juxtaglomerular apparatus during embryonal and fetal development.

Thirty kidneys from nine embryos, 20 fetuses, and one full-term baby were examined for their renin content by immunofluorescence and the peroxidase antiperoxidase method, using an antihuman renin antiserum. Renin-containing cells were found in the early metanephros (5-week-old fetuses). Most of them were located in the wall of well-developed renal arteries in the vicinity of the prospective vascular pole of the glomeruli. In the poorly differentiated peripheral renal cortex, intracellular fluorescence was seen in nearby arterioles of pocket-like s-shaped tubules. Rarely, labeled cells were found in the wall of major branches of renal arteries. In all locations, the renin-containing cells appear to be clearly linked to the development of the renal vascular system.