Anti-renin Antibody Research Articles

102. AAV9 Exclusively Targets the Juxtaglomerular Apparatus in the Adult Mouse Kidney After Intravenous Injection

Aim: Gene delivery to the kidney by adeno-associated virus (AAV) remains a relatively unexplored field. We found that the cells in renal tissue infected by AAV9 vectors were very few but interestingly distributed in some specific regions adjacent to the glomerulus. Therefore we aim to identify the type of the cells infected by the AAV9 vectors, and compare it with other AAVs for the specific targeting by different routes of vector injection.Material and Methods: We injected AAV9-CMV-GFP, AAVH43-CMV-GFP and AAVH48-CMV-GFP (5×1011 vg/mouse) into the adult C57BL/6 mice (n=4 /group) via the tail vein. For the renal parenchyma injection (n=3/group) the vectors (10μl, 1 × 1011vg) were slowly injected into the renal parenchyma by a 33-gauge needle, which was connected to a 50μl Hamilton syringe with PE10 tubing. Two weeks later, kidneys were frozen and sectioned to slices of 10 μm.Results: We found that the AAV9 vector specifically infected juxtaglomerular apparatus in the kidney by intravenous injection. The infection efficiency reached up to 58.2±3.2% of total juxtaglomerular apparatus. On the other hand, The AAVH43 and AAVH48 vectors were negative in the kidney yet achieved GFP expression similarly to AAV9 in the heart and liver. Immunostaining showed the cells infected by AAV9-CMV-GFP vectors were exclusively adjacent to juxtaglomerular (JG) cells. These JG cells synthesize, store and secrete renin, and were identified by anti-renin antibody. Neuronal nitric oxide synthase (nNOS) and prostaglandin E receptor 3 (EP3) immunostaining further indicated that GFP-positive cells were peri-macular cells, including the cells opposite the macula densa plaque. These were epithelial cells around the macula densa and one of four contact sites between the distal nephron and vasculature. The peri-macular cells, not including the macula densa, can generate spontaneous [Ca2+ ]i oscillations, and signal to the connected arterioles, regulating renal blood flow and tubular flow. Interestingly, specific infection in the juxtaglomerular apparatus by AAV9 vectors was dependent on the route of vector injection. When injected directly into the renal parenchyma, the peri-macular cells could no longer be infected, whereas the AAVH43 and AAVH48 vectors infected renal tubular epithelial cells.Conclusion: We have identified for the first time that a peri-macular cell type of the kidney is selectively targeted by intravenous AAV9 injection. Considering the importance role of those cells in regulation of renal blood flow and tubular flow, specific targeting by AAV9 may present a novel therapeutic method for renal hypertension and renal dysfunction. On the other hand, the data provide novel proof that the peri-macular cells are a group of functional epithelial cells, which differ from other renal tubular cells and may highly express AAV9 receptors. Additionally, the specificity of AAV9 infection in peri-macular cells was via the blood vessel, suggesting unique communication routes between peri-macular cells and the arterioles.

Accumulation of renin and imidazolone in peritubular capillary endothelial cells in insulin-resistant hypertensive rats

Peritubular endothelium plays a key role in the development and progression of diabetic and nondiabetic chronic kidney disease. Renal injury in disorders of glucose metabolism may appear as early as in the stage of impaired glucose tolerance (IGT) and is accelerated by hypertension. The aim of our study was to investigate renal histology in rats with hypertension and IGT, with special emphasis on the peritubular endothelium. Hypertension and IGT (H/IGT) were provoked in adult male Wistar rats by bilateral administration of methylglyoxal into the ventromedial hypothalamus. Immunohistochemistry with anti-renin and anti-imidazolone antibodies and immunoelectron microscopy with anti-renin antibody were performed. H/IGT rats showed tubulointerstitial fibrosis as well as renin and imidazolone staining in the papillary region. The patterns of immunostaining for renin and imidazolone were similar to that of endothelium. On electron microscopy, peritubular capillary endothelial cells and to a less extent, tubular epithelial cells showed renin positivity. Impaired glucose tolerance complicated with hypertension leads to tubulointerstitial fibrosis in rat kidney. Imidazolone deposition and renin production in peritubular capillary endothelial cells may play a role in the development of tubulointerstitial fibrosis.

Cardiac hypertrophy in vitamin D receptor knockout mice: role of the systemic and cardiac renin-angiotensin systems.

Our recent studies suggest that 1,25-dihydroxyvitamin D3 functions as an endocrine suppressor of renin biosynthesis. Genetic disruption of the vitamin D receptor (VDR) results in overstimulation of the renin-angiotensin system (RAS), leading to high blood pressure and cardiac hypertrophy. Consistent with the higher heart-to-body weight ratio, the size of left ventricular cardiomyocytes in VDR knockout (KO) mice was markedly increased compared with wild-type (WT) mice. As expected, levels of atrial natriuretic peptide (ANP) mRNA and circulating ANP were also increased in VDRKO mice. Treatment of VDRKO mice with captopril reduced cardiac hypertrophy and normalized ANP expression. To investigate the role of the cardiac RAS in the development of cardiac hypertrophy, the expression of renin, angiotensinogen, and AT-1a receptor in the heart was examined by real-time RT-PCR and immunostaining. In VDRKO mice, the cardiac renin mRNA level was significantly increased, and this increase was further amplified by captopril treatment. Consistently, intense immunostaining was detected in the left ventricle of captopril-treated WT and VDRKO mice by use of an anti-renin antibody. Levels of cardiac angiotensinogen and AT-1a receptor mRNAs were unchanged in the mutant mice. These data suggest that the cardiac hypertrophy seen in VDRKO mice is a consequence of activation of both the systemic and cardiac RAS and support the notion that 1,25-dihydroxyvitamin D(3) regulates cardiac functions, at least in part, through the RAS.

Characterization of renin activity in brown adipose tissue.

Angiotensin (ANG) II plays a vital role in blood pressure regulation and body fluid homeostasis. Although many peripheral tissues synthesize components of the renin-ANG system, very few synthesize all of the major components involved in the generation ofANG II. This study used interscapular brown adipose tissue (ISBAT) as a model system to evaluate the mechanism of ANG II generation in an extrarenal tissue. Polymerase chain reaction analysis of DNA from ISBAT demonstrated angiotensinogen gene expression; however, renin gene expression was not detected. Renin activity that was not completely derived from the residual blood pool was detected in ISBAT homogenates. Kinetic parameters for renin activity were similar in ISBAT and adrenal gland. Renin activity was partially inhibited by anti-renin antibody and completely inhibited by a specific rat renin inhibitor. Bilateral nephrectomy did not decrease renin activity in ISBAT. Western blot analysis, employing two species-specific renin antibodies, indicated the presence of a variety of isoforms of renin in ISBAT. The presence of renin activity in isolated brown adipocytes demonstrated that the enzyme is localized to adipocytes. The release of immunoreactive ANG peptides from ISBAT slices over 3 h indicated de novo synthesis. These studies support the existence of a local renin-ANG system in ISBAT and suggest involvement of renin in the formation of ANG II.

Renin immunochemistry, sodium excretion and relative heart weight in cyclosporine- or alimentary-induced magnesium deficiency in rats.

Rats were given a magnesium-(Mg) depleted (Mgd), or a Mg-standard (Mgst) or a Mg-enriched (Mge) diet, with 20 mg/kg/day cyclosporine (Cy) or olive oil per os for 90 days (6 groups). Anti-renin antibody was applied and the percent of renin-positive glomeruli (RI) was taken. Sodium excretion (NaU), relative heart weight (HW), as a measure of hypertension, and total femur Mg were measured. Compared to dietary controls, femur Mg was reduced under Cy and Mgd or Mgst indicating Mg deficiency. RI was higher in all Cy groups (p < 0.01), and Nau was lower in Mgd + Cy and in Mgst + Cy (p < 0.01). Correspondingly, HW was found to be significantly higher in Mgd + Cy and Mgst + Cy. In animals under Mge + Cy, there were no differences in NaU and HW compared to controls. The results indicate a relation between Cy-related hypertension and Mg status: Mg deficiency seems to enhance the hypertensive effect of Cy via sodium retention.

Deleterious effects of inhibition of the renin-angiotensin system in neonatal rats.

The angiotensin I converting enzyme inhibitor (ACEI) perindopril (2 mg/kg body weight), the peripheral vasodilator dihydralazine (DHL) (25 mg/kg body weight) or distilled water was given daily from birth to day 14 to neonatal rats. Blood pressure, plasma creatinine, plasma renin activity (PRA), substrate (PRS) and concentration (PRC) and renin content of kidney tissue sections were evaluated on days 14 and 28. By day 14, a high mortality in the ACEI group was observed. ACEI, but not DHL, led to a significant fall (P < 0.01) in blood pressure, 57 +/- 11 versus 89 +/- 25 in the DHL group and 103 +/- 24 mmHg in controls, and to a dramatic increase in plasma creatinine. PRA and PRS were undetectable in ACEI-treated rats; in contrast, PRC and renal staining with anti-renin antibody were significantly increased in ACEI rats. On day 28, the blood pressure was normal in all groups and plasma creatinine returned to the normal range in ACEI rats. PRA, PRS and PRC were not significantly different in the ACEI group and controls. These results suggest that the renin-angiotensin system (RAS) plays a major postnatal role in the neonatal rat. Inhibition of the RAS during the first 2 weeks of life leads to high mortality, severe hypotension, reversible renal failure and a defect in circulating angiotensinogen.

Role of renal nerves for the expression of renin in adult rat kidney

Utilizing a combination of mechanical and chemical unilateral denervation, we have examined the relevance of renal innervation for the expression of renin in kidneys of adult rats. Renal denervation led to a reduction by 57 +/- 4% of renin-containing areas in denervated kidneys as quantitated by morphometry of kidney sections immunoreactive against a polyclonal antirenin antibody. Preprorenin mRNA content in the denervated kidneys fell to 46 +/- 7% of the contralateral innervated kidneys. Treatment of rats with the beta 1-adrenoreceptor antagonist metoprolol (100 mg.kg-1.day-1) for 2 days decreased renal renin mRNA levels to 71% of control levels. Unilateral renal denervation led to a further decrease of renin mRNA levels also in metoprolol-treated animals to 60% of the values found in the contralateral kidneys. Hypotensive hemorrhage led to a 1.4-fold increase of renin mRNA in the kidneys of sham-treated animals. In unilaterally denervated rats renin mRNA increased to levels similar to those in sham-operated animals in both denervated and in contralateral innervated kidneys in response to bleeding. As a consequence, the ratio of abundance of renin mRNA in the denervated to the innervated kidneys rose to 86 +/- 7%. Pretreatment of the animals with metoprolol, on the other hand, prevented the rise of renin mRNA in response to hypotensive hemorrhage. Our findings suggest that in the adult organism renal neural input significantly contributes to the expression of renin under basal conditions, while it appears to be of less importance for stimulation of renin gene expression by severe blood loss.

Anti-renin T cells trigger normal B cells to produce anti-renin antibodies and normalize blood pressure in spontaneously hypertensive rats.

Spontaneously hypertensive (SH) rats immunized with mouse renin produce anti-renin antibodies, responsible for down-modulation of blood pressure, associated with an infiltration of kidneys by mononuclear cells. In this work, anti-renin T cells from SH rats immunized with renin have been stimulated in vitro, and we have studied in vitro and in vivo their effect on anti-renin antibody production by normal syngeneic B cells. We show that, in vitro, renin-activated T cells induce a renin-specific antibody response without addition of exogenous renin. Anti-renin T cells injected into naive SH rats trigger normal B cells to secrete high amounts of monospecific anti-renin IgG antibodies as early as day 5. These antibodies interfere with the homeostasis of the renin-angiotensin system leading to the normalization of blood pressure without any nephritis. These results show that anti-renin B cells are either not tolerant per se or in a reversible state of anergy. Our results also suggest that anti-renin B cells constitutively express renin-derived peptides in such a way that they may be stimulated by activated anti-renin T cells; these cells express IL-4 mRNA indicating that IL-4 could play a role in the differentiation of B cells.

Morphological and immunohistochemical characteristics of dimethylnitrosamine-induced malignant mesenchymal renal tumor in F-344 rats

Mesenchymal renal tumors in F-344 newborn rats were induced by a single dose of dimethylnitrosamine. The induced tumors were successfully transplanted into adult rats under the renal capsule. Neither the primary nor the transplanted neoplasms from various generations of grafts changed their morphological features during the tumor passage, having the same cellularity with high mitotic activity and the tendency to invade the host kidney rapidly. On the basis of lectin histochemistry and immunohistology, the tumor proved to be a mesenchymal neoplasm without any obvious capacity of the proliferating cells to differentiate into any well-known organoid element normally found in mature renal parenchyma. However, the proliferating neoplastic cells were found to have a strong vimentin positivity with desmin expression. Ultrastructurally, myofilaments with attachment bodies characteristic of smooth muscle cells were generally present in various amounts in many tumor cells. In addition, on the basis of the physiological data and on kidney/tumor renin activity obtained, it is interesting to note that the tumor-graft-invaded kidneys retained their enzyme activity, despite the obvious loss of renal tissue including glomeruli. However, the immunohistochemical findings with anti-renin antibody have clearly shown that this is not due to a renin-producing tumor but rather to the surviving (probably) non-neoplastic arterioles retaining the capacity to produce renin. Although these arterioles have mostly been found next to necrotic areas, commonly occurring in dimethylnitrosamine-induced transplantable renal tumors, the question of a possible physiological role of renin in tumor necrosis or in angiogenesis has remained open.

Conversion to renin of exogenously administered recombinant human prorenin in liver and kidney of monkeys

Highly purified recombinant human prorenin, labeled with 125I (125I-prorenin), was intravenously given to monkeys to examine the possible in vivo conversion of this prorenin to renin. 125I-prorenin and 125I-renin were detected using specific anti-prorenin prosegment antibody and anti-renin antibody, respectively. The plasma disappearance of immunoreactive 125I-prorenin in marmosets showed two exponential components with a half-life of 10.4 +/- 0.2 min for the rapid component and 165.7 +/- 12.6 min for the slow component. Fifteen minutes after the injection of 125I-prorenin, 38.7 +/- 2.8 and 3.9 +/- 0.5% of the administered dose accumulated in the liver and kidney, respectively. Less than 1% of the dose injected distributed in the other organs, including the brain, submandibular gland, lung, heart, aorta, adrenal gland, spleen, uterus, ovary, and testis. Thus the labeled prorenin was predominantly taken up by the liver and kidney. Analysis of liver and kidney extracts and plasma, by both gel permeation high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, demonstrated that 125I-prorenin (Mr = 46,000) taken up by the liver and kidney was significantly converted to 125I-renin (Mr = 42,000), whereas only a negligible amount of 125I-renin (Mr = 42,000) was present in the plasma. Although there seems to be no activation of prorenin in the blood circulation, prorenin does seem to be activated by the liver and kidney.

Interaction between the vascular renin-angiotensin system and prostaglandins.

The effects of nephrectomy, prostacyclin (PGI2), prostaglandin E2 (PGE2) and indomethacin on the vascular renin-angiotensin system were examined using isolated perfused mesenteric arteries. Angiotensin II (Ang II) released from the vasculature was measured using a Sep-Pak C18 cartridge which was placed in the perfusion system. After perfusion with drugs, the specific vascular renin activity inhibited by antirenin antibody was determined. Plasma renin activity was markedly decreased 48 h after nephrectomy, whereas vascular renin activity was increased. Released Ang II from mesenteric arteries of nephrectomized rats was not significantly different from that in control rats. Infusion of PGI2 (10(-6) mol/l) and PGE2 (10(-6) mol/l) for 1 h caused significant decreases in Ang II release (P less than 0.01 and P less than 0.05, respectively) and also decreased vascular renin activity. In contrast, infusion of indomethacin (10(-6) mol/l) for 1 h resulted in an increase (P less than 0.01) in vascular renin activity. These findings suggest that the vascular renin-angiotensin system exists independently of the circulating renin-angiotensin system. In contrast with their effects on renal renin, prostaglandins suppress the vascular renin-angiotensin system. The interaction between two vasoactive hormones in the vascular wall may be important for local regulation of vascular tone and regional blood flow.

Renin in human fetal lung--a biochemical and immunohistochemical study.

Human fetal lung homogenates contain an inactive form of renin which may be revealed by trypsin treatment. When activated, this form of renin has some biochemical similarities with fetal kidney renin: the pH optimum of fetal lung renin is approximately 6.5; it is bound by Affigel Blue affinity chromatography resin; and is inhibited by a monoclonal antibody (R-3-36-16) raised to human kidney renin. Inactive renin, partially-purified from both fetal kidney and lung, differs from this in that the renal form is of low-molecular weight (LMW, 45,000 daltons), whereas that from fetal lung is of high molecular weight (HMW, 58,000 daltons). Using a sensitive alkaline phosphatase-anti-alkaline phosphatase (APAAP) procedure with a polyclonal anti-renin antibody (R-15), immunoreactive renin in fetal lung was found in vessels in mesenchyme between airways. The pattern of staining was distributed similarly to Factor VIII-related antigen, suggesting localization in endothelial cells.

Effect of vasodilator prostaglandins on the vascular renin-angiotensin system

The interaction of prostaglandin (PG) with the vascular renin-angiotensin (R-A) system was examined by studies on the effects of PGI 2, PGE 2 and the inhibitor of PG synthesis, indomethacin, on the release of angiotensin II (Ang II) from isolated rat mesenteric arteries. The Ang II released from the vasculature was measured after its concentration in a Sep-Pak C 18 cartridge connected to the perfusion system. After perfusion with drugs, the specific vascular renin activity inhibited by anti-renin antibody was determined. The basal perfusion pressure was constant (19.6±1.1 mmHg at a flow rate of 4.5 ml/min, and was not changed by any of these drugs. The basal levels of Ang II release and vascular renin activity were 44 ± 5 pg/30 min and 113 ± 8 pg Ang I/mg protein /hr, respectively. Infusion of PGI 2 (10 −6 M) significantly decreased both Ang II release (p<0.01) and vascular renin activity (p<0.05) as compared with the control levels. Infusion of PGE 2 (10 −6 M) decreased Ang II release significantly (p<0.05) and vascular renin activity slightly. Infusion of indomethacin (10 −6M) increased vascular renin activity significantly (p<0.01). Pretreatment with indomethacin (10 mg/kg, ip) for 2 days also increased vascular renin activity (p<0.01). These results indicate that in contrast to their effects on the renal R-A system, PGs suppress the vascular R-A system and that these two local vasoactive factors interact to regulate vascular tone.

Multiple forms of immunoreactive renin in human adrenocortical tumour tissue from patients with primary aldosteronism.

There is increasing evidence which suggests that the adrenal gland contains the renin-angiotensin cycle. The localization of renin has been reported to be mainly in the zona glomerulosa rather than the fasciculata medullary portion. In the present study we have investigated extracts from aldosteronomas (n = 3), which are believed to derive from the zona glomerulosa cells. In addition, we have attempted to characterize the biochemical properties of the adrenal renin. Sizable quantities of renin-like activity (32.0 +/- 7.7 ng of angiotensin I generated h-1 mg-1 of protein, mean +/- SEM) were detected in the extracts. This renin-like activity was inhibited by anti-renin antibody raised against pure renin (mean, 95% of the total renin-like activity), indicating that it was not due to the non-specific action of proteases such as cathepsin D. The optimum pH of the tissue renin-like enzyme was 6.0 for rat plasma substrate. Differences were found, however, in the molecular mass (36,000, 37,000, 44,000 and 48,000), binding to concanavalin A and isoelectric points (4.40, 4.68 and 5.00). These results confirm the existence of specific renin in aldosteronoma. Renin microheterogeneity could be evidence for local production of the enzyme.

Hypotensive Effect of a Human Anti-Renin Monoclonal Antibody (4G1D8) in the Sodium Depleted Conscious Marmoset

Hypotensive Effect of a Human Anti-Renin Monoclonal Antibody (4G1D8) in the Sodium Depleted Conscious Marmoset

A Monoclonal Antibody Specific for the Amino Terminal Sequence of Human Prorenin Identifies a Common Epitope on Renal and Amniotic Fluid Inactive Renins

A synthetic nonapeptide corresponding to the predicted amino terminus of human prorenin was synthesized and used as an antigen in the production of mouse hybridomas. Forty-seven clones producing antibody to this peptide were identified and screened for ability to bind to partially purified amniotic fluid inactive renin in a soluble phase assay. None showed activity. One purified antibody, 4D3-3C4, was found to bind by Western blotting to renal and amniotic fluid inactive renins, molecular size 52 kDa, after gel electrophoresis in sodium dodecyl sulphate (SDS) following reduction with mercaptoethanol. In contrast, the anti-renin monoclonal antibody, R3-27-6, recognizes active renin, molecular size 38 kDa, and two inactive renin species, 42 kDa and 52 kDa, in the same renin preparations. Kidney and amniotic fluid apparently contain an inactive renin that possesses the complete prorenin sequence. These data indicate that high molecular weight inactive renin from both kidney and amniotic fluid contain an epitope, accessible to antibody only after denaturation, which represents the amino-terminal octapeptide sequence of prorenin. The inactive renin preparations tested also contain smaller fragments which either possess this epitope or do not, while still reacting with antibodies to active renin. It is likely that all these smaller inactive renin species are the result of proteolysis, either post-synthetic or in vitro.

Induction of Renin Activity by Gonadotropic Hormones in Cultured Leydig Tumor Cells*

The hormonal regulation of renin activity in cloned and cultured Leydig tumor cells (designated MA-10) was examined. The treatment of Leydig cell cultures with bovine LH (bLH), hCG, or with (Bu)2cAMP elicited a dose- and time-dependent induction of renin activity and a concomitant increase in steroid biosynthesis. The optimum concentration of hCG was 25 ng/ml, which caused an average 25-fold increase in renin activity compared to the control value. bLH action was optimum at 75-100 ng/ml and induced an approximately 35-fold increase in renin activity. The maximum inducible level of renin activity was attained after 8-9 h of hormone treatments. The addition of progesterone (the major steroid product of the MA-10 cells) did not induce a significant increase in renin activity. Treatment of MA-10 cells with epidermal growth factor also failed to produce any increase in renin activity. The optimum concentration of (Bu)2cAMP was 800 microM for the induction of renin activity and caused an approximately 40-fold increase compared to the control value. Renin activity induced by bLH, hCG, or (Bu)2cAMP was completely inhibited by mouse anti-renin antibody, indicating the specific nature of renin. Upon withdrawal of (Bu)2cAMP from the culture medium, renin activity gradually declined to the control level, and with retreatment of these cultures with (Bu)2cAMP, a newly induced state of enzyme activity was resumed. Indirectly, the role of new protein and RNA synthesis was examined during hormonal regulation of renin induction using protein and RNA synthesis inhibitors such as cycloheximide, puromycin, actinomycin D, or rifampicin. Both protein and RNA synthesis inhibitors blocked the induction of renin activity in the presence of all three inducing agents, bLH, hCG, or (Bu)2cAMP. The results provide evidence that the induction of renin activity is modulated by bLH, hCG, or (Bu)2cAMP and represent the de novo synthesis of enzyme molecules.

Renin and angiotensins in cultured mouse adrenocortical tumour cells

Mouse adrenal tissue has been reported to contain high renin activity. However, it is not clear whether the renin is produced inside the tissue or is derived from a blood-borne component. We have investigated a cloned cell line of mouse adrenocortical tumour (Y-1) which has a steroidogenic activity. Sizable quantities of renin were demonstrated, predominantly in the cell lysate. This renin activity was distinguished from cathepsin D in view of its specific affinity to anti-renin antibody, optimal pH was determined, and the substrate specificity was checked with haemoglobin. Immunoreactive angiotensins were also detectable, but were demonstrated both in the cell and in the culture medium. This study provides further evidence for the existence of renin intrinsic to the adrenal gland. This study also suggests an intracellular role for renin and possible secretion of generated angiotensins.

Biochemical properties of renin in human pituitary tissue

The biochemical properties of renin, extracted from human pituitary specimens obtained at autopsy, were studied using a specific antirenin antibody raised against human kidney renin. The following results were obtained. The molecular weight of pituitary renin was estimated to be about 37,000 daltons by gel filtration through Sephadex G-100. The optimum pH of pituitary renin was between 6.0 approximately 7.0, while that of a renin-like substance which did not react with the antirenin antibody had an acidic pH of 4.0, with a pH comparable to that of the cathepsin D-like enzyme in the pituitary tissue. The presence of two different isoelectric-point species of pituitary renin was revealed by isoelectric focusing, one with a point of pH 4.47 and the other with that of pH 5.77. The Km value of pituitary renin was 37.9 microM for synthetic human renin substrate. Affinity chromatography of the pituitary renin on a Concanavalin-Sepharose column showed that most (87.4%) of the pituitary renin did not contain glycoprotein residues. Treatment with either trypsin or glandular kallikrein increased the renin activity, indicating the presence of an inactive form of renin in the pituitary tissue. From these findings, it is concluded that specific renin exists in human pituitary tissue. It seems likely that the pituitary renin is of local origin rather than contamination of the circulating enzyme.

Immunoreactive renin in mouse adrenal gland. Localization in the inner cortical region.

The existence of renin in the adrenal gland of the mouse was determined by its enzymatic activity and by immunohistochemical techniques using monospecific antibodies to mouse submandibular gland renin. The adrenal gland of mouse was found to contain a very high level of renin significantly greater than other mouse tissues except for the kidney and submandibular gland. Also, the renin level in mouse adrenal was significantly higher than that in adrenals of other species. This renin activity was distinct from the nonspecific renin-like activity of acid proteases in that its activity was optimal at neutral pH and specifically inhibited by antirenin antibody. Adrenal renin increased upon nephrectomy indicating that it is not derived from the kidney. Immunohistochemical studies localized the renin-immunoreactive substance to cells in the inner region of the cortex. The intensity of staining was highest in the innermost region and decreased in cells in outer layers.