Juxtaglomerular Cell Granules Research Articles

Phenotypic dissection of the mouse Ren1d knockout by complementation with human renin

Normal renin synthesis and secretion is important for the maintenance of juxtaglomerular apparatus architecture. Mice lacking a functional Ren1d gene are devoid of renal juxtaglomerular cell granules and exhibit an altered macula densa morphology. Due to the species-specificity of renin activity, transgenic mice are ideal models for experimentally investigating and manipulating expression patterns of the human renin gene in a native cellular environment without confounding renin–angiotensin system interactions. A 55-kb transgene encompassing the human renin locus was crossed onto the mouse Ren1d-null background, restoring granulation in juxtaglomerular cells. Correct processing of human renin in dense core granules was confirmed by immunogold labeling. After stimulation of the renin–angiotensin system, juxtaglomerular cells contained rhomboid protogranules with paracrystalline contents, dilated rough endoplasmic reticulum, and electron-lucent granular structures. However, complementation of Ren1d−/− mice with human renin was unable to rescue the abnormality seen in macula densa structure. The juxtaglomerular apparatus was still able to respond to tubuloglomerular feedback in isolated perfused juxtaglomerular apparatus preparations, although minor differences in glomerular tuft contractility and macula densa cell calcium handling were observed. This study reveals that the human renin protein is able to complement the mouse Ren1d−/− non-granulated defect and suggests that granulopoiesis requires a structural motif that is conserved between the mouse Ren1d and human renin proteins. It also suggests that the altered macula densa phenotype is related to the activity of the renin-1d enzyme in a local juxtaglomerular renin–angiotensin system.

Abstract 108: Imaging Renin Granule Exocytosis by Total Internal Reflection (TIRF) Microscopy in Mouse Juxtaglomerular Cells: Effect of cAMP and Beta Adrenergic Stimulation

Renin is stored in dense core granules in juxtaglomerular (JG) cells. Renin release is highly regulated. However, the kinetics of renin release and mode of granule exocytosis remains unexplored. We developed a new method for real-time visualization of renin granule exocytosis by TIRF microcopy. We hypothesize that renin exocytosis is due to a kiss-and-run mode of exocytosis, where fusion of the granule with the plasma membrane is transient and granule integrity is maintained. To study this, we generated a new adenoviral construct encoding full length mouse renin tagged with Carboxyl-terminus yellow fluorescent protein (Ad-Renin-YFP). First, we characterized its expression and activity in an endocrine pituitary cell line that does not express endogenous renin (Att20 cells). By Western Blot, we observed a band at the expected molecular mass of renin-YFP (70 kDa)(n=4). Ad-Renin-YFP retains its enzymatic activity in Att20 cells since angiotensin I conversion from angiotensinogen was only detected in Att20 cells transduced with renin-YFP (n=3; p <0.01). To monitor exocytosis of renin-YFP, we transduced primary cultures of mouse JG cells and monitored granule movement and their changes in fluorescence intensity within 250 nm of the plasma membrane by TIRF microscopy. Under baseline conditions the average number of docked granules was 16±4 granules per cell (n=12). Translational movement (X-Y planes) of docked granules was negligible. The number of events per cell during 10 minutes was low and no full fusion was detected (1.5 ±0.5 total events per cell, n=4). After stimulation with cAMP, the number and frequency of events increased to 5.3±1.0 events per cell/10 min (n=8, p<0.05). Similarly the number of events increased to 4.25±0.9 when JG cells were stimulated with isoproterenol, without a decrease in the number of granules docked (n=9). While most (73.5%) of the exocytic events occurred from docked granules; only 36.5% of events were caused by recruitment of newcomer granules to the TIRF field. We conclude that in JG cells, full fusion of granules is not the main mechanism of renin exocytosis. The rapid bursts in fluorescence intensity of docked granules suggest that kiss-and-run is the main mechanism of stimulated-renin exocytosis, in a highly regulated process.

Bimodal dynamics of granular organelles in primary renin-expressing cells revealed using TIRF microscopy.

Renin is the initiator and rate-limiting factor in the renin-angiotensin blood pressure regulation system. Although renin is not exclusively produced in the kidney, in nonmurine species the synthesis and secretion of the active circulatory enzyme is confined almost exclusively to the dense core granules of juxtaglomerular (JG) cells, where prorenin is processed and stored for release via a regulated pathway. Despite its importance, the structural organization and regulation of granules within these cells is not well understood, in part due to the difficulty in culturing primary JG cells in vitro and the lack of appropriate cell lines. We have streamlined the isolation and culture of primary renin-expressing cells suitable for high-speed, high-resolution live imaging using a Percoll gradient-based procedure to purify cells from RenGFP+ transgenic mice. Fibronectin-coated glass coverslips proved optimal for the adhesion of renin-expressing cells and facilitated live cell imaging at the plasma membrane of primary renin cells using total internal reflection fluorescence microscopy (TIRFM). To obtain quantitative data on intracellular function, we stained mixed granule and lysosome populations with Lysotracker Red and stimulated cells using 100 nM isoproterenol. Analysis of membrane-proximal acidic granular organelle dynamics and behavior within renin-expressing cells revealed the existence of two populations of granular organelles with distinct functional responses following isoproterenol stimulation. The application of high-resolution techniques for imaging JG and other specialized kidney cells provides new opportunities for investigating renal cell biology.

VAMP8 knockout mice show enhanced renin release and content, kidney damage and salt sensitivity (860.5)

The vesicle fusion SNARE protein VAMP8 mediates trafficking of aquaporin 2 in kidney collecting ducts. Gene deletion of VAMP8 in mice (V8‐KO) resulted in polyuria and enhanced plasma renin. Renin is stored in granules in juxtaglomerular (JG) cells and its release is mediated by SNARE proteins. In other endocrine cells, VAMP8 mediates granule maturation and constitutive exocytosis. We found that VAMP8 is expressed in JG cells. However, it is not known whether VAMP8 regulates renin release from JG cells or the enhanced plasma renin in V8‐KO is due to compensation for volume loss. We hypothesized that VAMP8 mediates renin granule maturation and constitutive renin release from JG cells. Primary cultures of JG cells from V8‐KO showed enhanced renin and pro‐renin protein expression compared to wild type (WT) (n=5). Baseline renin release from JG cells was 5‐fold higher in V8‐KO than WT (WT=0.06±0.01; KO=0.3±0.05 μgAngI/hr/mg prot.; p<0.05). Immunolabeling of renin and confocal imaging showed enlarged granules and vacuoles in JG cells from V8‐KO. Similar results were observed in V8‐KO JG cells by electron microscopy. Kidney histology showed enhanced renal fibrosis and glomerular sclerosis in V8‐KO (n=4). Despite the higher release of renin and kidney damage, baseline blood pressure in V8‐KO was similar to WT (mmHg: WT=122±3.4; V8‐KO=121±4.5). A high salt diet (1% NaCl drinking water) increased blood pressure in V8‐KO mice by 16±5 mmHg (p<0.05) but not in age matched WT controls. We concluded that VAMP8 plays an inhibitory role in renin release, most likely by mediating normal granule maturation. VAMP8 may regulate blood pressure by inhibiting renin release during a high salt diet. VAMP8 is protective against kidney damage, but the mechanism is unclear.Grant Funding Source: Supported by AHA and NIH

Procollagen I-expressing renin cell precursors

Renin-expressing cells in the kidney normally appear as mural cells of developing preglomerular vessels and finally impose as granulated juxtaglomerular cells in adult kidneys. The differentiation of renin-expressing cells from the metanephric mesenchyme in general and the potential role of special precursor stages in particular is not well understood. Therefore, it was the aim of this study to search for renin cell precursors in the kidney. As an experimental model, we used kidneys of aldosterone synthase-deficient mice, which display a prominent compensatory overproduction of renin cells that are arranged in multilayered perivascular cell clusters. We found that the perivascular cell clusters contained two apparently distinct cell types, one staining positive for renin and another one staining positive for type I procollagen (PC1). It appeared as if PC1 and renin expression were inversely related at the cellular level. The proportion of renin-positive to PC1-positive cells in the clusters was inversely linked to the rate of salt intake, as was overall renin expression. Our findings suggest that the cells in the perivascular cell clusters can reversibly switch between PC1 and renin expression and that PC1-expressing cells might be precursors of renin cells. A few of those PC1-positive cells were found also in adult wild-type kidneys in the juxtaglomerular lacis cell area, in which renin expression can be induced on demand.

Complementation of juxtaglomerular cell granulation in Ren‐ 1d null mice by human renin

Mice null for the Ren‐1d gene lack renin storage granules in their juxtaglomerular cells and exhibit altered macula densa morphology. Three lines of transgenic mice carrying a 55kbp transgene encompassing the human renin (hRen) locus were generated and crossed onto a Ren1d‐null background. Expression of hRen was seen to restore granulation in juxtaglomerular (JG) cells in a transgene expression level‐dependent manner, suggesting that a threshold level of renin expression is required for the formation of dense renin‐containing granules. Complementation of the phenotype by the human enzyme suggests that hRen and mouse Ren1d share conserved epitopes required for trafficking renin into the regulated secretion pathway and hence for granulopoiesis. To dissect the granulation phenotype in detail, 2D electron microscopic (EM) images were taken of kidney sections, which were then reconstructed in 3D to show the full extent of renin granular structure. As expected, the expression of hRen, which does not effectively cleave mouse angiotensinogen, was unable to restore macula densa morphology, indicating that a locally active RAS is required for normal macular densa structure and function.

Role of the SNARE protein SNAP23 on cAMP-stimulated renin release in mouse juxtaglomerular cells

Renin, the rate-limiting enzyme in the formation of angiotensin II, is synthesized and stored in granules in juxtaglomerular (JG) cells. Therefore, the controlled mechanism involved in renin release is essential for the regulation of blood pressure. Exocytosis of renin-containing granules is likely involved in renin release; a process stimulated by cAMP. We found that the "soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor" (SNARE) protein VAMP2 mediates cAMP-stimulated renin release and exocytosis in JG cells. To mediate exocytosis, VAMP2 must interact with a synaptosome-associated protein (SNAP). In the renal cortex, the isoform SNAP23 is abundantly expressed. We hypothesized that SNAP23 mediates cAMP-stimulated renin release from primary cultures of mouse JG cells. We found that SNAP23 protein is expressed and colocalized with renin-containing granules in primary cultures of mouse JG cell lysates. Thus, we then tested the involvement of SNAP23 in cAMP-stimulated renin release by transducing JG cells with a dominant-negative SNAP23 construct. In control JG cells transduced with a scrambled sequence, increasing cAMP stimulated renin release from 1.3 ± 0.3 to 5.3 ± 1.2% of renin content. In cells transduced with dominant-negative SNAP23, cAMP increased renin from 1.0 ± 0.1 to 3.0 ± 0.6% of renin content, a 50% blockade. Botulinum toxin E, which cleaves and inactivates SNAP23, reduced cAMP-stimulated renin release by 42 ± 17%. Finally, adenovirus-mediated silencing of SNAP23 significantly blocked cAMP-stimulated renin release by 50 ± 13%. We concluded that the SNARE protein SNAP23 mediates cAMP-stimulated renin release. These data show that renin release is a SNARE-dependent process.

The effect of aminoguanidine on the kidney of diabetic albino Balb/c mice

The aim of this study is to find out how activated inducible nitric oxide synthase (iNOS) and nitric oxide (NO) affect kidney tissue in streptozotocin (STZ)-induced diabetic mice and whether its influence can be prevented by aminoguanidine (AG), a specific iNOS inhibitor. Twenty-four male mice were divided into four study groups (n=6) receiving a daily dose of 100 mg.kg-1 AG for 90 days (Group AG), a single dose of 150 mg.kg-1 STZ (Group STZ), a single dose of 150 mg.kg-1 STZ followed by daily administration of 100 mg.kg-1 AG for 90 days (Group STZ-AG), and intraperitoneally injections of saline only (Group Control) for 90 days. Dispersion of NADPH-diaphorase (NADPH-d) was stronger in the kidney sections of STZ-treated animals compared with the controls. STZ treatment caused disruption of continuity of the brush borders in proximal tubules, glomerular endothelial damage, and considerable renin granules in the juxtaglomerular cells. AG administration following STZ treatment partly prevented histological and cytological changes in kidney cortex, and renin dispersion was similar to that in control animals. We found that increased inducible nitric oxide (iNO) caused kidney tissue degeneration that could be prevented to some extent by AG treatment. There is a possible relationship between increased iNOS and dispersion of renin granules in juxtaglomerular cells in diabetes.

Enhanced renin release and content in juxtaglomerular (JG) cells from VAMP8 knockout mice

Renin is essential for angiotensin II production and blood pressure control. Renin is stored in granules in juxtaglomerular (JG) cells. We showed that vesicle associated membrane protein 2 (VAMP2), a member of SNARE family, mediates stimulated renin exocytosis in JG cells. However, the VAMP isoform required for constitutive renin release is unknown. In other cells VAMP8 mediates granule maturation and constitutive exocytosis. We hypothesized that VAMP8 mediates constitutive renin release in JG cells. We used primary cultures of JG cells from wild type (WT) and VAMP8 knockout (V8‐KO) mice and measured VAMP8 localization and renin released to the medium during 1 hr. In wild type, VAMP8 was expressed in JG cell granules. Baseline renin release was 5‐fold higher in V8‐KO than WT (WT=0.06±0.01; KO=0.3±0.05 μgAngI/hr/mg prot.; p<0.05). Total renin content was 5‐fold higher in V8‐KO than WT (WT=2.9±0.2; KO=16.7±0.6 μgAngI/hr/mg prot.; p<0.05). The fraction of total renin released to the media was similar in V8‐KO and WT JG cells (WT: 1.6±0.3; KO: 1.7±0.5% of total renin) indicating higher number of granules in V8‐KO, or higher renin content/granule. Immunolabeling of renin and confocal imaging showed enlarged granules and vacuoles in JG cells from V8‐KO. We concluded that: 1) VAMP8 does not mediate constitutive renin exocytosis; and 2) VAMP8 may play an inhibitory role in transcription, translation or processing of renin. 5F32HL096346

Vesicle-associated Membrane Protein-2 (VAMP2) Mediates cAMP-stimulated Renin Release in Mouse Juxtaglomerular Cells

Renin is essential for blood pressure control. Renin is stored in granules in juxtaglomerular (JG) cells, located in the pole of the renal afferent arterioles. The second messenger cAMP stimulates renin release. However, it is unclear whether fusion and exocytosis of renin-containing granules is involved. In addition, the role of the fusion proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins), in renin release from JG cells has not been studied. The vesicle SNARE proteins VAMP2 (vesicle associated membrane protein 2) and VAMP3 mediate cAMP-stimulated exocytosis in other endocrine cells. Thus, we hypothesized that VAMP2 and/or -3 mediate cAMP-stimulated renin release from JG cells. By fluorescence-activated cell sorting, we isolated JG cells expressing green fluorescent protein and compared the relative abundance of VAMP2/3 in JG cells versus total mouse kidney mRNA by quantitative PCR. We found that VAMP2 and VAMP3 mRNA are expressed and enriched in JG cells. Confocal imaging of primary cultures of JG cells showed that VAMP2 (but not VAMP3) co-localized with renin-containing granules. Cleavage of VAMP2 and VAMP3 with tetanus toxin blocked cAMP-stimulated renin release from JG cells by ∼50% and impaired cAMP-stimulated exocytosis by ∼50%, as monitored with FM1–43. Then we specifically knocked down VAMP2 or VAMP3 by adenoviral-mediated delivery of short hairpin silencing RNA. We found that silencing VAMP2 blocked cAMP-induced renin release by ∼50%. In contrast, silencing VAMP3 had no effect on basal or cAMP-stimulated renin release. We conclude that VAMP2 and VAMP3 are expressed in JG cells, but only VAMP2 is targeted to renin-containing granules and mediates the stimulatory effect of cAMP on renin exocytosis.

Role of the SNARE protein SNAP‐23 on cAMP‐stimulated Renin release from mouse Juxtaglomerular (JG) cells

Exocytosis of renin containing granules is likely involved in renin release from juxtaglomerular (JG) cells; a process stimulated by cAMP. In non‐neuronal endocrine cells granule exocytosis is mediated by SNAP‐23, a protein of the soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) family. We hypothesized that cAMP‐stimulated renin release from JG cells is in part mediated by SNAP‐23. We measured: 1) the expression of SNAP‐23 by Western blot and immunofluorescence confocal microscopy; and 2) the effect of adenoviral delivery of a dominant‐negative SNAP‐23 and botulinum toxin E on cAMP‐stimulated renin release in mouse JG cell primary cultures. cAMP was increased with Forkolin plus IBMX (F/I). SNAP‐23 was detected by Western blot in JG cell lysates and co‐localized with renin granules in JG cells. In control JG cells transduced with a scramble sequence, increasing cAMP stimulated renin release from 1.3±0.3 to 5.3±1.2 % of renin content. In cells transduced with dominant‐negative SNAP‐23, F/I increased renin from 1.0±0.1 to 3±0.6 % of renin content, a 50% blockade (p<0.02). Botulinum toxin E, which cleaves and inactivates SNAP‐23, reduced cAMP‐stimulated renin release from 5.0±0.7 to 2.45±0.5 % of renin content, a 50% blockade (p< 0.05). We concluded that the SNARE protein SNAP‐23 partially mediates cAMP‐stimulated renin release. These data show for the first time that renin release is mediated by SNAREs.

Cathepsin B is not the processing enzyme for mouse prorenin

Renin, an aspartyl protease that catalyzes the rate-limiting step in the renin-angiotensin system (RAS), is proteolytically activated by a second protease [referred to as the prorenin processing enzyme (PPE)] before its secretion from the juxtaglomerular cells of the kidney. Although several enzymes are capable of activating renin in vitro, the leading candidate for the PPE in the kidney is cathepsin B (CTSB) due to is colocalization with the renin precursor (prorenin) in juxtaglomerular cell granules and because of its site-selective activation of human prorenin both in vitro and in transfected tissue culture cell models. To verify the role of CTSB in prorenin processing in vivo, we tested the ability of CTSB-deficient (CTSB-/-) mice to generate active renin. CTSB-/- mice do not exhibit any overt symptoms (renal malformation, preweaning mortality) typical of an RAS deficiency and have normal levels of circulating active renin, which, like those in control animals, rise more than 15-fold in response to pharmacologic inhibition of the RAS. The mature renin enzyme detected in kidney lysates of CTSB-/- mice migrates at the same apparent molecular weight as that in control mice, and the processing to active renin is not affected by chloroquine treatment of the animals. Finally, the distribution and morphology of renin-producing cells in the kidney is normal in CTSB-/- mice. In conclusion, CTSB-deficient mice exhibit no differences compared with controls in their ability to generate active renin, and our results do not support CTSB as the PPE in mice.

Granulation Rescue and Developmental Marking of Juxtaglomerular Cells Using “Piggy-BAC” Recombination of the Mouse RenLocus

Mice lacking a functional Ren-1(d) gene exhibit a complete lack of renal juxtaglomerular cell granulation and atypical macula densa morphology. Transgenic mice carrying a 145-kilobase BAC clone encompassing the Ren-1(d) and Ren-2 loci were generated, characterized, and backcrossed with Ren-1(d-/-) mice. Homozygous Ren-1(d)-null mice expressing the BAC clone exhibited complete restoration of normal renal structure. Homologous recombination in Escherichia coli was used to generate a modified version of the BAC clone, in which an IRESbeta-geo cassette was inserted specifically into the Ren-1(d) gene. When introduced into the germline, the modified clone provided a marker for juxtaglomerular cell differentiation and beta-geo was expressed appropriately in juxtaglomerular cells throughout development. Parallel backcross experiments onto the Ren-1(d)-null background demonstrated that the juxtaglomerular cells expressed the modified Ren-1(d) locus in the absence of regranulation. These data demonstrate that the nongranulated cells constitute bona fide juxtaglomerular cells despite their altered morphology, that overexpression of renin-2 cannot compensate for the loss of renin-1(d), and that primary structural differences between the two isoforms are responsible for the differences in granulation. The use of BAC modification as part of functional complementation studies illustrates the potential for in vivo molecular dissection of key physiological mechanisms.

Renin-1 is essential for normal renal juxtaglomerular cell granulation and macula densa morphology.

The secretion of renin from granules stored in renal juxtaglomerular cells plays a key role in blood pressure homeostasis. The synthesis and release of renin and the extent of granulation is regulated by several mechanisms including signaling from the macula densa, neuronal input, and blood pressure. Through the use of a gene-targeting vector containing homology arms generated using the polymerase chain reaction, we have inactivated the Ren-1(d) gene, one of two mouse genes encoding renin, and report that lack of renin-1(d) results in altered morphology of the macula densa of the kidney distal tubule and complete absence of juxtaglomerular cell granulation. Furthermore, Ren-1(d-/-) mice exhibit sexually dimorphic hypotension. The altered growth morphology of the macula densa in Ren-1(d)-null mice should provide a tool for the investigation of the JG cell-macula densa signaling. Furthermore, the current data indicate that expression of the Ren-1(d) gene is a prerequisite for the formation of storage granules, even though the related protein renin-2 is present in these mice, suggesting that renin-1(d) and renin-2 are secreted by distinct pathways in vivo.

Exocytosis of secretory granules in the juxtaglomerular granular cells of kidneys

There is little agreement as to the secretory process of renin granules in juxtaglomerular granular cells (JG cells) of kidneys, although a large number of studies of the regulation of renin secretion have been reported. The structural correlation between the stimuli and the secretory process was examined in mouse JG cells on renal cortical slices incubated with the beta-adrenergic agonist, isoproterenol; the loop diuretic, furocemide; the Ca2+ chelator, EGTA; and the actin filament-disrupting agent, cytochalasin B. Treatment with isoproterenol (10(-5)-10(-3) M) or furocemide (10(-3) M) in Ca(2+)-containing medium did not significantly affect the ultrastructure of JG cells. In slices incubated with isoproterenol or furocemide in the Ca(2+)-free medium, JG cells occasionally contained a few electron-lucent granules at the cell periphery in addition to the electron-dense mature granules observed in the control slices. On rare occasions, the JG cells displayed omega-shaped cavities with electron-lucent matrices, a feature similar to the contents of electron-lucent granules. Cytochalasin B markedly promoted the effects of these stimulants in Ca(2+)-free medium. These findings suggest that participation of actin filament disassembly in the exocytotic process of the mature granules in JG cells.

Histopathological and ultrastructural changes in the juxtaglomerular apparatus of the rat following administration of ZENECA ZD6888 (2-ethyl-5,6,7,8-tetrahydro-4-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)- methoxy]quinoline), an angiotensin II antagonist.

Male and female Alderley Park rats were treated for 26 wk with ZENECA ZD6888, an angiotensin II (AII) antagonist, at doses of 25, 50, and 250 mg/kg/day and the kidneys examined by light and electron microscopy. Nephropathy (tubular degeneration and regeneration), thickening of the basal lamina, mononuclear cell infiltration, and hypertrophy and hyperplasia of the juxtaglomerular apparatus were seen by light microscopy. Ultrastructurally, juxtaglomerular cell granules were increased in number and displayed substantial structural heterogeneity. In some cells, granules contained paracrystalline inclusions while others consisted predominantly of myelinic debris. Evidence of conversion of arteriolar smooth muscle cells into juxtaglomerular cells and their degeneration was also obtained by electron microscopy. The juxtaglomerular changes were treatment- and dose-related and were attributed to an exaggerated pharmacological action of the compound, that is, ZD6888-mediated blockade of AII receptors leading to competitive inhibition of the AII-mediated release of renin. The resemblance between the findings seen in the present study following administration of an AII antagonist and those due to angiotensin-converting enzyme inhibitors reported by others was striking.

Role of cathepsin B as prorenin processing enzyme in human kidney.

Renin is synthesized from an inactive precursor, prorenin, through cleavage at a pair of basic amino acids catalyzed by prorenin processing enzyme (PPE). A lysosomal protease, cathepsin B, has been suggested to be a strong candidate for PPE. However, there still remains a possibility that other protease(s) can also catalyze prorenin processing. We studied the subcellular distribution of PPE in human renal cortex using pure recombinant prorenin as a PPE assay substrate. PPE and renin activities, and cathepsin B activity and protein were colocalized in the lysosomal fraction. The PPE activity was completely inhibited by a cathepsin B specific inhibitor, CA074. Taken together with the immunohistochemical data showing that cathepsin B and PPE are colocalized in dense secretory granules of juxtaglomerular cells of kidney, we conclude that cathepsin B is the authentic PPE in human kidney.

Immunocytochemical localization of cathepsin B in rat anterior pituitary endocrine cells, with special reference to its co-localization with renin and prorenin in gonadotrophs.

We examined by immunocytochemistry the localization of cathepsin B in endocrine cells of rat anterior pituitary lobe, using a monospecific antibody to cathepsin B. By light microscopy, granular immunodeposits for cathepsin B were detected in most endocrine cells of anterior pituitary lobe. Cells immunoreactive for luteinizing hormone (LH) were diffusely immunostained by anti-cathepsin B. By electron microscopy, immunogold particles for cathepsin B were localized in lysosomes of thyrotrophs, somatotrophs, and mammotrophs. In mammotrophs, immunogold particles for cathepsin B were also detected in crinophagic bodies. Double immunostaining co-localized immunogold particles for LH and cathepsin B in secretory granules of gonadotrophs. Immunocytochemistry was also applied to demonstrate localization of renin and prorenin in LH-producing gonadotrophs; immunogold particles for renin were co-localized with those for LH, cathepsin B, or prorenin in their secretory granules. Immunogold particles for prorenin were also co-localized with those for LH or cathepsin B in secretory granules, but prorenin-positive granules appeared less frequently than renin-positive granules. These results suggest that cathepsin B not only plays a role in the protein degradation in lysosomes of anterior pituitary endocrine cells but also participates in the activation of renin in gonadotrophs, as has been demonstrated in secretory granules of juxtaglomerular cells.

Use of conventional embedment procedures for post-embedding immunocytochemistry of secretory proteins

The methodology of post-embedding immunocytochemistry commonly employs regimens that are more technically difficult than conventional processing and staining of specimens for transmission electron microscopy. Among these regimens are the preparation of frozen thin sections and embedment in hydrophilic acrylic resins such as Lowicryl and LR White . Cryotechnology, however, requires special equipment and exacting preparation of specimens. Use of water-soluble resins likewise demands special care; for example, embedment in LR White involves titrating the dehydration solutions and maintaining oxygen-free curing conditions (usually in gelatin capsules, making selected orientation of specimens quite difficult). It is commonly supposed that conventional treatments such as exposure to osmium tetroxide and uranyl acetate en bloc, with subsequent embedment in epoxy resins, are detrimental to antigenicity.Uranyl acetate block-staining can actually enhance immunocytocheinical staining of some epitopes . Though osmicated tissues have also been successfully immunostained , it is standard procedure first to treat the thin sections on grid with an oxidizing agent such sodium metaperiodate . We have found that a polyclonal antibody to renin effectively immunostains globular inclusions (granules) in juxtaglomerular (JG) cells of rat kidney. This can be accomplished both in unosmicated tissue embedded in LR White (Fig. 1) or in specimens (either intact kidney or isolated JG cells) that have been osmicated, uranyl acetate block- stained, dehydrated through 100% ethanol, and embedded in Poly/Bed epoxy resin (cured at 60 °C). Thin sections were collected on nickel grids and immunostained with Protein A-gold, without prior oxidation to remove osmium (Fig. 1). There are several distinct advantages of using the latter immunocytochemical procedure. First, tissues can be processed in a routine manner; in addition, specific orientation of specimens can easily be accomplished. Furthermore, curing of the embedment does not have to be carried out at low temperatures or in oxygen-free containers; this is particularly useful when processing cultured cells (Fig. 1).

Correlation of 24-hour fluctuations in renin granules of juxtaglomerular cells and in renin and angiotensinogen in blood plasma of the rat.

Subcellular structures of juxtaglomerular (JG) cells in the rat kidney were morphometrically examined at six evenly spaced times over 24 h. Plasma renin activities and angiotensinogen concentrations were also measured at these times. The cell volumes were larger at 20.00 h and 04.00 h than at 00.00 h, whereas the nuclear volumes peaked at 20.00 h and 08.00 h, decreasing at 00.00 h and 16.00 h. The volume and surface densities of renin granules and their individual volumes and surface areas peaked at 16.00 h and 00.00 h, decreasing at 20.00 h and 08.00 h, whereas their numerical densities peaked at 20.00 h, decreasing at 12.00 h. The surface densities of the rough endoplasmic reticulum (rER) peaked at 20.00 h, decreasing at other times, except at 08.00 h, when rER volume and surface density were relatively high. The plasma renin activity was maximal at 20.00 h, whereas it was minimal at 08.00 h. The variation in plasma angiotensinogen concentrations was inversely correlated with that in plasma renin activities. These results suggest that JG cells actively synthesize and release renin during the dark period, especially at 20.00 h, whereas during the light period they gradually synthesize renin and produce the granules, most of which may be stored in the cells during this period.