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Structural analysis suggests that renin is released by compound exocytosis
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http://www.kidney-international.org & 2012 International Society of Nephrology
Structural analysis suggests that renin is released by compound exocytosis Dominik Steppan1, Anita Zu¨gner1, Reinhard Rachel2 and Armin Kurtz1 1
Physiologisches Institut der Universita¨t Regensburg, Regensburg, Germany and 2Anatomisches Institut der Universita¨t Regensburg, Regensburg, Germany
The mode of renin release from renal juxtaglomerular cells into circulation is still unsolved in several aspects. Here we studied the intracellular organization of renin-storage vesicles and their changes during controlled stimulation of renin release. This was accomplished using isolated perfused mouse kidneys with 3-dimensional electron microscopic analyses of renin-producing cells. Renin was found to be stored in a network of single granules and cavern-like structures, and dependent on the synthesis of glycosylated prorenin. Acute stimulation of renin release led to increased exocytosis in combination with intracellular fusion of vesicles to larger caverns and their subsequent emptying. Renin release from the kidneys of SCID-beige mice, which contain few but gigantic renin-storage vesicles, was no different from that of kidneys from wild-type mice. Thus, our findings suggest that renin is released by mechanisms similar to compound exocytosis. Kidney International (2013) 83, 233–241; doi:10.1038/ki.2012.392; published online 12 December 2012 KEYWORDS: afferent arteriole; angiotensin; renin–angiotensin system
Correspondence: Armin Kurtz, Physiologisches Institut der Universita¨t Regensburg, D-93053 Regensburg, Germany. E-mail: [email protected] Received 8 August 2012; revised 10 September 2012; accepted 13 September 2012; published online 12 December 2012 Kidney International (2013) 83, 233–241
The protease renin is the key enzyme regulating the activity of the renin–angiotensin–aldosterone system.1 It is mainly produced and secreted by specialized kidney cells located in the terminal parts of afferent arterioles.2,3 In these cells, renin is synthesized as a precursor protein prorenin, which is glycosylated in the Golgi apparatus and then transferred into storage vesicles.1,4–6 Within these storage vesicles, prorenin is proteolytically processed to renin.5 The release of renin from the cells is mainly controlled by two oppositely acting signaling pathways. The cyclic adenosine monophosphate (cAMP) signaling pathway stimulates renin release, whereas a calcium-related signaling pathway inhibits the release of renin.7–10 Inhibition of secretion by calcium is unusual, as calcium is considered to be essential for the induction or maintainance of secretory events, in particular of exocytosis.11 In fact, morphological signs of exocytosis, such as omega-shaped figures, have very rarely been reported in renin-producing cells,12,13 in spite of the numerous and prominent renin-containing vesicles, which often show an irregular shape at the ultrastructural level.14,15 Therefore, it has been hypothesized that the release of renin from storage vesicles may occur through intracellular solubilization of renin, which then is transported through the plasma membrane.16,17 Functional data such as a quantlike release of renin,18 increase of membrane capacitance19,20, and the disappearance of renin-storage vesicles upon stimulation of renin release,21 however, would support the idea of a controlled exocytosis of renin. Others have described membrane invaginations in the course of renin release.22–24 Signs of exocytosis of renin-containing vesicles such as omega-shaped membrane structures have been occasionally described for kidneys of animals, in which renin synthesis and secretion had been stimulated in vivo by adrenalectomy in combination with treatment with the loop diurectics.12 Notably, no signs of exocytosis have been reported for animals chronically treated with angiotensinconverting-enzyme (ACE) inhibitors,22 although the number of renin-storage vesicles clearly decreases during acute ACE-I treatment.22,25 In addition, periods of acute renal hypoperfusion lead to a decrease of the renin-storage vesicle number without morphological signs of exocytosis.22 Finally, beige mice lacking the Lyst protein produce only very few but huge renin-storage vesicles, without signs of 233
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RESULTS
Renin secretion rates of normal mouse kidneys perfused under standard conditions reached stable renin release rates after 15 min (Figure 1). After a control perfusion period of 5 min, kidneys were perfusion-fixed for electron microscopic analysis. Renin-producing cells in the juxtaglomerular areas contained numerous electron-dense vesicles that frequently showed an irregular circumference on 2D sections (Figure 2a). Up to 100 serial sections per cell were prepared to generate a 3D picture of renin-storing vesicles. These 3D reconstructions revealed that different forms of vesicles existed, ranging from single granules to huge interconnected caverns (Figure 2b and c). We analyzed a total of six cells from three kidneys, which all displayed a rather similar appearance. The distribution of single granules and caverns showed some variability even among renin-producing cells of the same kidneys under control conditions, suggesting that granules and cisternae are states of a dynamic equilibrium (Figure 2d). Common to all of these cells was that the vesicles covered around 36% of the extranuclear space of the cells (Table 1). The average volume of a vesicle was around 0.6 fl (Table 1). In six individual cells under control conditions, we reconstructed up to 50% of the cell surfaces. However, we did 234
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exocytosis.26 The plasma renin concentration reflecting secreted renin, is lower in these mice, however, in proportion with a decreased number of renin-expressing cells in the kidneys. Therefore, renin release from individual reninproducing cells may not be very different between wild-type and beige mice. Experiments with a more defined stimulation of renin release have been performed with incubated kidney slices, which were incubated with isoprenaline, calcium chelators, furosemide, cytochalasin B, or combinations of these compounds.12,13 Again, morphological signs of exocytosis were very rarely seen. More evident was the appearance of electron-lucent and/or empty vesicles, which were considered as indirect signs for exocytosis.12,13 Notably, isoproterenol, which is a powerful and immediately acting stimulator of renin release, did not induce morphological changes of renin-secreting cells.13 It was the combination of the calcium chelator ethylene glycol tetraacetic acid (EGTA) with cytochalasin B that produced a partial emptying of renin-storage vesicles within 20 min of incubation.13 However, it is not clear as to whether the combination of these nonphysiological compounds might have induced effects that do not reflect the physiological release mode of renin. In summary, the mode of renin release is still rather mysterious in several aspects. For a better understanding of this process, we were therefore interested to learn more about the morphology of renin-storage vesicles and their possible changes during controlled modulation of renin release. For this goal, we combined the model of the isolated perfused mouse kidney, which allows us to modulate renin release under quasi physiological conditions with three-dimensional (3D) electron microscopy of renin-producing cells in the kidney.
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Figure 1 | Perfusion protocol and renin secretion rates of isolated wild-type mouse kidneys. After a stabilization period of 15 min, the samples were taken for the determination of basal renin release (control period C). For determination of the normal juxtaglomerular cell structure, kidneys were fixed at the end of the control period (time point 1). After the control period, isoproterenol (ISO, 5 nmol/l) was added to the perfusate. Five minutes later, kidneys were fixed for electron microscopical (ELMI) analysis (time point 2). Further, during isoproterenol infusion, ethylene glycol tetraacetic acid (EGTA) (2.5 m) was added to the perfusate to lower the extracellular concentration of calcium. At 5 min after the start of EGTA infusion, kidneys were fixed for ELMI analysis (time point 3). Renin secretion data are means±s.e.m. of nine kidneys for the control period, six kidneys for the period during ISO infusion only, and three kidneys after the start of EGTA infusion. AngI, angiotensin I.
not observe membrane figures indicative for exocytosis, such as omega-shaped structures. We also did not observe electron-lucent or empty vesicles in these cells. In parallel, we also perfused and analyzed kidneys that expressed the ren-2, but not the ren-1d, gene. In the perfusate of these kidneys, very low enzymatic renin activity (5 ng AngI/hmlg) could be measured, which, however, was not increased by isoproterenol, nor by lowering of calcium. In juxtaglomerular cells of these kidneys, electron-dense interconnected vesicular structures could be seen as well (Figure 3a–c). The average volume of these vesicles in ren-2 kidneys was clearly smaller than those found in ren-1d/ren-2 kidneys (Table 1). Moreover, the total vesicular volume in ren-2 kidneys was less than 20% of those measured in ren-1d/ ren-2 kidneys (Table 1). We next analyzed cells from ren-1d/ren-2 kidneys in which renin secretion was prestimulated by isoproterenol (5 nmol/l) for 5 min, which led to a fivefold increase of renin secretion from the isolated kidneys (Figure 1). In none of the six cells analyzed did we find signs of exocytosis or structural alterations of the vesicular renin-storage system (not shown). During isoproterenol infusion (5 nmol/l), we then lowered the extracellular concentration of calcium (by the addition of EGTA 2.5 mmol/l) for another 5 min. This maneuver led to a 20-fold stimulation of renin release over basal values (Figure 1). Now, rearrangment of the vesicles became apparent on 2D sections (Figure 4a). Larger caverns due to Kidney International (2013) 83, 233–241
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Table 1 | Average vesicle volumes and total vesicular volumes of juxtaglomerular cells Mouse strain and kidney perfusion protocol
Average vesicle volume (fl)
Volume ratio (vesicles/extranuclear cell volume) (%)
ren-1d þ / þ (WT) control ren-2 þ / þ control ren-1d þ / þ (WT) (5 min ISO þ 5 min ISO þ EGTA) ren-1d þ / þ (WT) (15 min ISO þ EGTA) SCID-Beige control
0.6±0.05 0.25±0.09* 1.9±0.3*
35.7±3.2 6±1* 40.5±2.9
2.5±0.3*
36.4±3.5
4.4±0.2*
40±3.9
Abbreviations: EGTA, ethylene glycol tetraacetic acid; ISO, isoproterenol; SCID, severe combined immunodeficiency; WT, wild type. Data are means±s.e.m. of 6 cells each. *Po0.05 versus ren-1d wild-type control.
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Figure 2 | Analysis of a wild-type juxtaglomerular cell after control perfusion according to the protocol shown in Figure 1. (a) Transmission electron microscopy section of the juxtaglomerular cell (original magnification 3800) shows vesicles of different sizes and forms. (b–d) 3D reconstruction of the cell with individual renin vesicles in different colors and the nucleus in brown color. Vesicle structure ranged from single granules (b) to interconnected caverns (c). The intracellular arrangement of vesicles/caverns is shown in d.
fusion of smaller vesicles developed (Figure 4b and c), and the average volume of the storage vesicle increased from 0.6 to 1.9 fl (Table 1). The proportion of total vesicle volume over extranuclear space was not changed (Table 1). In addition, distinct exocytoses became visible, which did not show preferential localization to the endothelial or to the adventitial site. Such excytoses resulted from single granules, but also from extensions of larger cisternae (Figure 4d). Prolonged strong stimulation with isoproterenol in the presence of low extracellular calcium concentration for 15 min (Figure 5) led to exocytoses and to marked changes of vesicle appearance (Figure 6a). Apart from vesicles with normal appearance, numerous vesicles with lower electron density showed up. Electron-lucent and empty vesicles appeared, which were mostly fused to one vesicular complex (Figure 6b). Some vesicles appeared as emptied structures. As a consequence of the fusion of the vesicles, the average volume of the storage vesicles increased further to 2.5 fl, but the proportion of total vesicle volume over extranuclear space remained unchanged (Table 1). Kidney International (2013) 83, 233–241
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Figure 3 | Analysis of a juxtaglomerular cell of a ren-2-expressing mouse after control perfusion according to the protocol shown in Figure 1. (a) Transmission electron microscopy section of the juxtaglomerular cell (original magnification 3800) shows a few irregularly shaped electron-dense vesicles. (b, c) 3D reconstruction of the cell, with renin vesicles shown in dark gray. Vesicles appear to be interconnected, forming cavern-like structures (c).
The sum of the findings obtained so far would indicate that during stimulation of renin release intracellular vesicle fusion rather than vesicle fission occurs, suggesting that vesicle fission is not required for the stimulation of renin secretion. To test for this conclusion, we analyzed renin secretion from kidneys of severe combined immunodeficiency (SCID)-beige mice that carry a defective Lyst-like protein. The Lyst-like protein is relevant for vesicle fission.27 Basal renin release from beige kidneys was not different from that of wild-type kidneys (Figure 7). Transmission electron microscopy (TEM) analysis of renin cells of beige kidneys revealed a few, huge vesicular structures (Figure 8a). 3D analysis confirmed the existence of a few but huge caverns 235
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Figure 5 | Perfusion protocol and renin secretion rates of isolated wild-type mouse kidneys. After a stabilization period of 15 min, samples were taken for the determination of basal renin release (control period C). After the control period, isoproterenol (ISO, 5 nmol/l) in combination with ethylene glycol tetraacetic acid (EGTA) (2.5 mmol/l) was added to the perfusate. After 15 min, kidneys were fixed for ELMI analysis (time point F). Renin secretion data are means±s.e.m. of three kidneys. AngI, angiotensin I.
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Figure 4 | Analysis of a wild-type juxtaglomerular cell after subsequent perfusion with isoproterenol and ethylene glycol tetraacetic acid (EGTA) according to the protocol shown in Figure 1. (a) Transmission electron microscopy section of the juxtaglomerular cell (original magnification 3800) shows numerous irregularly shaped vesicles. (b, c) 3D reconstruction of the cell with individual renin vesicles in different colors and the nucleus in brown color. Intracellular caverns developed, which were larger than single vesicles. Distinct caverns are indicated by different colors. (d) Distinct exocytoses became visible. These exocytoses resulted from single granules, but also from extensions of larger caverns.
and few additional single granules (Figure 8b–d). The average volume of the vesicles was clearly increased (Table 1). Notably, the proportion of total vesicle volume over extranuclear volume space was similar to that of normal mice (Table 1). Under basal perfusion conditions, we did not notice signs of exocytosis in six cells of three kidneys examined. Stimulation of renin secretion by isoproterenol and by lowering of the extracellular concentration calcium for 5 min each produced renin secretion rates that were not clearly different between beige mice and normal mice (Figure 7). Both analysis of TEM images (Figure 9a) and 3D analysis (Figure 9b and c) of these cells revealed the appearance of exocytoses but no obvious signs of a rearrangement of the storage structures. The fusion of vesicles with the plasma membrane or intracellular fusion of vesicles requires physical contact between these structures. As our data did not provide 236
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Figure 6 | Analysis of a wild-type juxtaglomerular cell after perfusion with isoproterenol and ethylene glycol tetraacetic acid according to the protocol shown in Figure 5. (a) Transmission electron microscopy section of the juxtaglomerular cell (original magnification 3800). According to their electron densities, four populations of storage vesicles could be distinguished, namely vesicles with normal density (1), vesicles in transition from normal to lower density (2), vesicles with low density (electron-lucent) (3), and emptied vesicles (4). Electron-lucent and emptied vesicles are interconnected (indicated by arrowheads). (b) 3D reconstruction of the cell with the nucleus shown in medium-gray color. Apart from vesicles with normal appearance (dark gray), numerous vesicles with lower electron density became visible. Electron-lucent and emptied vesicles fused to huge caverns. One of these interconnected vesicle networks is depicted in light-gray color. Kidney International (2013) 83, 233–241
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D Steppan et al.: Renin release by compound exocytosis
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Figure 7 | Perfusion protocol and renin secretion rates of isolated severe combined immunodeficiency (SCID)-beige mouse kidneys. After a stabilization period of 15 min, the samples were taken for the determination of basal renin release (control period C). For the determination of the normal juxtaglomerular cell structure, kidneys were fixed at the end of the control period (time point 1). After the control period, isoproterenol (ISO, 5 nmol/l) was added to the perfusate. Then, during isoproterenol infusion, ethylene glycol tetraacetic acid (EGTA) (2.5 mmol/l) was added to the perfusate to lower the extracellular concentration of calcium. At 5 min after the start of EDTA infusion, kidneys were fixed for ELMI analysis (time point 2). For comparison, renin secretion rates of wild-type kidneys (taken from Figure 1) are also shown. Renin secretion data are means±s.e.m. of six kidneys for control period and three kidneys after control period. AngI, angiotensin I.
evidence for a directed movement of vesicles during the stimulation of renin release, we considered the possibility that the contact of the membranes may be controlled by the microfilament network.28–30 We therefore investigated the influence of compounds on renin secretion that are known to disrupt the microfilament network, such as cytochalasin D, which interrupts the actin–myosin microfilament network,31 or lantrunculin, which disrupts the F-actin network.32 However, neither compound added to the perfusate of isolated kidneys exerted an effect on basal renin secretion. In addition, there was no obvious effect of these compounds on the stimulation of renin secretion by isoproterenol (10 nmol/l), nor on the inhibitory effect exerted by angiotensin II (1 nmol/l) (Figure 10). DISCUSSION
Our data show that renin-storage vesicles do not show a uniform appearance such as typical secretory vesicles known from endocrine and exocrine cells.33,34 At the single-section electron microscopic level, renin-storage vesicles display irregular circumferences, which has already been observed by several investigators in the past.14,15,35 Our 3D reconstructions demonstrate that these irregularly shaped forms are 2D sections of different states of storage structures ranging from single vesicles to huge interconnected caverns. As the relative proportion of these different structures shows a significant variability between renin cells of otherwise Kidney International (2013) 83, 233–241
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Figure 8 | Analysis of a juxtaglomerular cell of a severe combined immunodeficiency (SCID)-beige mouse after control perfusion according to the protocol shown in Figure 7. (a) Transmission electron microscopy section of the juxtaglomerular cell (original magnification 3800) shows a few and huge irregularly shaped vesicles with inclusion bodies. (b–d) 3D reconstruction of the cell with individual renin vesicles in different colors and the nucleus in brown color. The form of the vesicles ranged from voluminous caverns (b) to flat and extended pancake-formed structures (c). The arrangement of the vesicles/caverns is shown in d.
untreated kidneys, we assume that the development of single vesicles and large cisternae might be a dynamic interchangeable and reversible process that involves fragmentation of caverns into vesicles and refusion of single vesicles into caverns. Our data confirm the previous notion that the development of big electron-dense granules in juxtaglomerular cells depends on the synthesis of the glycosylated prorenin.4,36 The nonglycosylated Ren-2 protein is constitutively released in its enzymatically inactive proform.37,38 Nonetheless, also ren-2 juxtaglomerular cells show some rudiments of electron-dense vesicular structures, probably reflecting the principal capability of the reninexpressing cells to develop such special structures. Our study also confirms previous reports on the relevance of the Lyst protein for the formation of smaller reninstorage vesicles.26 Apparently, the Lyst protein is essential for the segmentation of renin-storage cisternae into smaller vesicles. 237
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Figure 9 | Analysis of a juxtaglomerular cell of a severe combined immunodeficiency (SCID)-beige mouse after subsequent perfusion with isoporoterenol and ethylene glycol tetraacetic acid according to the protocol shown in Figure 7. (a) Transmission electron microscopy section of the juxtaglomerular cell (original magnification 3800) shows irregularly shaped vesicles. (b, c) 3D reconstruction of the cell with individual renin vesicles in different colors and the nucleus in brown color. There is no obvious rearrangement of the vesicles (b) relative to the nonstimulated state (Figure 8). Exoxytoses became apparent (c).
Our data now show that controlled stimulation of renin secretion by increasing the cAMP concentration and by lowering the calcium concentration leads to the appearance of morphological signs of exocytosis. A 20-fold stimulation of renin secretion was associated with an estimated number of five exocytotic events per cell, which were not seen under nonstimulated conditions. With a more moderate stimulation of renin secretion by isoproterenol alone, we did not see signs of exocytosis, which is also in accordance with data obtained from kidney slices.13 Although a number of five exocytoses per cells is small in comparison with other secretory cells, we think that the temporal coincidence between the stimulation of renin secretion and the appearance of exocytoses further supports the concept that exocytosis of storage vesicles is the relevant 238
Figure 10 | Renin secretion rates from wild-type mouse kidneys perfused with lantrunculin or cytochalasin D. After a stabilization period of 15 min, samples were taken for the determination of basal renin release (control period C). After the control period, either vehicle, lantrunculin, or cytochalasin D at concentrations of 3 mmol/l (period D1) or 10 mmol/l (period D2) were added to the perfusate. Renin secretion was stimulated by adding isoproterenol, 10 nmol/l (period ISO), or inhibited by adding angiotensin II, 1 nmol/l (period angiotensin II (Ang II)), to the perfusate. Renin secretion data are means±s.e.m. of three kidneys each.
pathway for the release of active renin.19–22 Given that the observed exocytoses account for the renin secretion during stimulation, one would extrapolate that, on average, among five cells, one might show one exocytosis to maintain basal renin secretion rates of the kidneys. It is not unlikely that such a low number of exocytoses under basal conditions would have escaped our notion. Such a low number of exocytoses would be in good agreement with calculations already made by Taugner et al.12 That profound changes of renin secretion can occur in spite of moderate numbers of exocytotic events likely results from the intracellular fusion of vesicles, which formed huge complex networks, that appeared to be emptied or contained electron-lucent material in states of stimulated secretion. One fusion pore with the plasma membrane thus allows the release of the content of several vesicles. Such an exocytotic release mode of stored material is termed compound exocytosis,39 which occurs in many cell types such as Kidney International (2013) 83, 233–241
D Steppan et al.: Renin release by compound exocytosis
pancreatic acinar cells, mast cells, eosinophils, and neutrophils.39–43 Compound exocytosis in combination with few extrusion pores in the plasma membrane likely explains the deep ‘plasma membrane invaginations’ that have been previously described in electron microscopic examinations of renin cells22–24 and which have already been speculated to reflect compound exocytosis. They become even more prominent during strong stimulation of renin release, which leads to an emptying of the cisternae. Apparently, vesicle fusion rather than vesicle fission occurs during the stimulation of renin secretion opposite to other endocrine cells. For example, in insulin-secreting b cells of the pancreas, stimulation of secretion causes an increased formation of single vesicles in the subplasmalemnal area.44–46 The conclusion on the relevance of intracellular vesicle fusion for renin secretion is also supported by the observation that renin secretion from kidneys that lack the Lyst protein, and thus show impaired formation of smaller vesicles, is similar to renin secretion from normal kidneys. This finding is in line with in vivo data indicating that plasma renin concentrations and the number of renin cells are reduced in proportion in beige mice,26 suggesting that renin secretion per cell is not very different between beige and wild-type mice. Our analysis further revealed that fusion of reninstorage vesicle with the plasma membrane is not restricted to smaller vesicles, but does also occur with large renincontaining cisternae as in beige mice. We did not obtain signs for a directed movement of vesicles in the direction of the plasma membrane during stimulation of secretion, as it occurs in other secretory cells.44,47 Instead, one could imagine that stimulation of renin secretion increases the fusogeneity of vesicle membranes once they get into contact with each other by random movement.48 The increase of fusogeneity of vesicle membranes could be induced by modifications of vesicle proteins, but also by modifications of the myofilament skeleton of the cells. As it had been reported that cytochalasin B favors exocytosis of renin from kidney slices, we investigated the effects of drugs disrupting actin filaments, such as cytochalasin D31 and latrunculin,32 on renin secretion from isolated perfused kidneys. However, we did not obtain evidence that any of the drugs interfered with renin secretion, suggesting that the actin filament has a minor role for the control of renin release. It cannot be excluded that the observed effect of cytochalasin B on renin release from kidney slices might result from side effects such as inhibition of glucose uptake,49 which might cause energy depletion in incubated kidney slices. It will be a task for future research to define the proteins relevant for fusion of renin-storage vesicles among themselves and with the plasma membrane. Obvious candidates for investigation would be members of the SNARE family.50,51 Our pilot experiments in this direction, however, indicate that the most common SNARE proteins found in endocrine cells are either lacking or are expressed at very low levels only in renin-secreting cells of the kidney. Kidney International (2013) 83, 233–241
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Therefore, future research should focus on unraveling the components of the exocytosis machinery in reninsecreting cells. MATERIALS AND METHODS Animals Experiments were normally conducted in 12- to 20-week-old male C57BL/6J mice (Jackson Laboratories). We also used male homozygous C57BL/6J-Lystbg-J/J (Beige) mice (Jackson Laboratories, Bar Harbor, Maine), which were clearly identifiable by their fur color. Homozygous Ren1d Cre/Cre (Ren-2) mice were generated by backcrossing of Ren1d þ /Cre- Ren-2 mice, which were kindly provided by Ariel Gomez, University of Virginia.52 For genotyping of the Ren1d Cre/Cre mice, tail biopsies were performed, and DNA was extracted and tested for the presence of wild-type and mutant genes using the following primers: Ren1d-sense, 50 -GAAGGAGAGC AAAAGGTAAGAG-30 ; Ren1d-antisense, 50 -GTAGTAGAAGGGGG AGTTGTG-30 ; and Cre, 50 -TTGGTGTACGGTCAGTAAATTGGAC30 . All animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health and were approved by the local ethics committee. Isolated perfused mouse kidney The isolated perfused mouse kidney model has been described in detail elsewhere.53 Briefly, the animals were anesthetized with an intraperitoneal injection of Ketamin (50 mg/kg body weight, Curamed, Karlsruhe, Germany) and Xylazin (80 mg/kg body weight, Ratiopharm, Ulm, Germany); the abdominal aorta was cannulated, and the right kidney was excised, placed in a thermostated moistening chamber, and perfused at constant pressure (90 mm Hg). Using an electronic feedback control, perfusion pressure could be changed and held constant in a pressure range between 40 and 140 mm Hg. Finally, the renal vein was cannulated and the venous effluent was collected for the determination of renin activity and venous blood flow. The basic perfusion medium consisted of a modified Krebs–Henseleit solution supplemented with 6 g/100 ml bovine serum albumin and with freshly washed human red blood cells (a 10% hematocrit). For the determination of renin secretion rates, three samples of the venous effluent were taken in intervals of 2 min during each experimental period. Renin activity in the venous effluent was determined by radioimmunoassay (Byk & DiaSorin Diagnostics, Seelze, Karlsruhe, Germany) as described elsewhere.53 Renin secretion rates were calculated as the product of the renin activity and the venous flow rate (ml/ming kidney weight). Renin secretion was modulated by adding defined concentrations of isoproterenol, EGTA, angiotensin II, latrunculin, or cytochalasin D (all from Sigma, Deisenhofen, Germany) to the perfusate. Fixation for transmission electron microscopy Kidneys were fixated with constant pressure (90 mm Hg) for 3 min by perfusion with phosphate-buffered saline (PBS) buffer containing 2% glutaraldehyde. The kidney was cut in half and stored at 4 1C in 2% glutaraldehyde/PBS until embedment for TEM. For any experimental condition, three kidneys were fixated. Embedment and transmission electron microscopy The kidney tissue was cut in 1-mm3-wide blocks and embedded in epoxyde resin (epoxy embedment kit, Fluka, Neu-Ulm, Germany) 239
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using an automatic microwave (Leica EM AMV, Leica, Germany). The embedded tissue was cut into 70-nm-thick serial slices using an ultramicrotome (EM UC7, Leica, Wetzlar, Germany), which were then placed on copper grids coated with pioloform. The serial slices were contrasted using a 4% uranyl acetate solution and a 0.5% lead citrate solution. For the acquisition of the images of a juxtaglomerular cell, a transmission electron microscope (Phillips CM12 TEM, Fei & Co, Eindhoven, Netherlands) with a LaB5 cathode and an acceleration voltage of 120 keV was used. The digitalization was carried out with a TEM-1000 slow-scan CCD camera and the program EM-Menu 4.0 (both from TVIPS-Tietz GmbH, Gauting, Germany). 3D tissue reconstruction We used a variation of the technique originally described by Sauter et al.54 The resulting serial images were converted to an image stack with constant picture size with the graphics software ImageJ. The image stack containing the sequence of B50–100 TEM images of a juxtaglomerular cell was then imported into the Amira 5.4.1 Visualization Software (Visage Imaging, Berlin, Germany). After correct scaling and alignment, various electron-dense structures of the cell were labeled and assigned to different defined materials. Finally, the surfaces were computed from the material data, resulting in a 3D model of the juxtaglomerular cell. For every experimental condition, two cells were reconstructed per kidney.
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DISCLOSURE
All the authors declared no competing interest. ACKNOWLEDGMENTS
The technical assistance provided by Marlies Hamann and Robert Go¨tz is gratefully acknowledged. We thank Frank Schweda for critical discussion. The study was financially supported by funds of the Deutsche Forschungsgemeinschaft (SFB 699).
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Barajas L. Anatomy of the juxtaglomerular apparatus. Am J Physiol 1979; 237: F333–F343. Taugner R, Metz R. Development and fate of the secretory granules of juxtaglomerular epithelioid cells. Cell Tissue Res 1986; 246: 595–606. King JA, Lush DJ, Fray JC. Regulation of renin processing and secretion: chemiosmotic control and novel secretory pathway. Am J Physiol 1993; 265: C305–C320. Inoue H, Takahashi S, Miyake Y. Modulation of active renin secretion by renin-binding protein (RnBP) in mouse pituitary AtT-20 cells transfected with human renin and RnBP cDNAs. J Biochem 1992; 111: 407–412. Skott O. Episodic release of renin from single isolated superfused rat afferent arterioles. Pflugers Arch 1986; 407: 41–45. Friis UG, Jensen BL, Aas JK et al. Direct demonstration of exocytosis and endocytosis in single mouse juxtaglomerular cells. Circ Res 1999; 84: 929–936. Friis UG, Jensen BL, Sethi S et al. Control of renin secretion from rat juxtaglomerular cells by cAMP-specific phosphodiesterases. Circ Res 2002; 90: 996–1003. Peti-Peterdi J, Fintha A, Fuson AL et al. Real-time imaging of renin release in vitro. Am J Physiol Renal Physiol 2004; 287: F329–F335. Rasch R, Jensen BL, Nyengaard JR et al. Quantitative changes in rat renin secretory granules after acute and chronic stimulation of the renin system. Cell Tissue Res 1998; 292: 563–571. Ryan GB, Alcorn D, Coghlan JP et al. Ultrastructural morphology of granule release from juxtaglomerular myoepithelioid and peripolar cells. Kidney Int Suppl 1982; 12: S3–S8. Peter S. Ultrastructural studies on the secretory process in the epithelioid cells of the juxtaglomerular apparatus. Cell Tissue Res 1976; 168: 45–53. Barrett GL, Morgan TO, Smith M et al. Effect of converting enzyme inhibition on renin synthesis and secretion in mice. Hypertension 1989; 14: 385–395. Jensen BL, Rasch R, Nyengaard JR et al. Giant renin secretory granules in beige mouse renal afferent arterioles. Cell Tissue Res 1997; 288: 399–406. Durchfort N, Verhoef S, Vaughn MB et al. The enlarged lysosomes in beige j cells result from decreased lysosome fission and not increased lysosome fusion. Traffic 2012; 13: 108–119. Kogel T, Gerdes HH. Maturation of secretory granules. Results Probl Cell Differ 2010; 50: 1–20. Caviston JP, Holzbaur EL. Microtubule motors at the intersection of trafficking and transport. Tr Cell Biol 2006; 16: 530–537. Aunis D, Bader MF. The cytoskeleton as a barrier to exocytosis in secretory cells. J Exp Biol 1988; 139: 253–266. Goddette DW, Frieden C. Actin polymerization. The mechanism of action of cytochalasin D. J Biol Chem 1986; 261: 15974–15980. Spector I, Shochet NR, Kashman Y et al. Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells. Science 1983; 219: 493–495. Burgoyne RD, Morgan A. Secretory granule exocytosis. Physiol Rev 2003; 83: 581–632. Castle JD, Cameron RS, Arvan P et al. Similarities and differences among neuroendocrine, exocrine, and endocytic vesicles. Ann N Y Acad Sci 1987; 493: 448–460. Dominick MA, Bobrowski WF, Metz AL et al. Ultrastructural juxtaglomerular cell changes in normotensive rats treated with quinapril, an inhibitor of angiotensin-converting enzyme. Toxicol Pathol 1990; 18: 396–406. Mullins LJ, Payne CM, Kotelevtseva N et al. Granulation rescue and developmental marking of juxtaglomerular cells using ‘piggy-BAC’ recombination of the mouse ren locus. J Biol Chem 2000; 275: 40378–40384. Clark AF, Sharp MG, Morley SD et al. Renin-1 is essential for normal renal juxtaglomerular cell granulation and macula densa morphology. J Biol Chem 1997; 272: 18185–18190. Sharp MG, Fettes D, Brooker G et al. Targeted inactivation of the Ren-2 gene in mice. Hypertension 1996; 28: 1126–1131. Scepek S, Lindau M. Focal exocytosis by eosinophils—compound exocytosis and cumulative fusion. EMBO J 1993; 12: 1811–1817. Bokvist K, Holmqvist M, Gromada J et al. Compound exocytosis in voltage-clamped mouse pancreatic beta-cells revealed by carbon fibre amperometry. Pflugers Arch 2000; 439: 634–645. Hafez I, Stolpe A, Lindau M. Compound exocytosis and cumulative fusion in eosinophils. J Biol Chem 2003; 278: 44921–44928. Lollike K, Lindau M, Calafat J et al. Compound exocytosis of granules in human neutrophils. J Leukoc Biol 2002; 71: 973–980. Kidney International (2013) 83, 233–241
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http://www.kidney-international.org & 2012 International Society of Nephrology
Structural analysis suggests that renin is released by compound exocytosis Dominik Steppan1, Anita Zu¨gner1, Reinhard Rachel2 and Armin Kurtz1 1
Physiologisches Institut der Universita¨t Regensburg, Regensburg, Germany and 2Anatomisches Institut der Universita¨t Regensburg, Regensburg, Germany
The mode of renin release from renal juxtaglomerular cells into circulation is still unsolved in several aspects. Here we studied the intracellular organization of renin-storage vesicles and their changes during controlled stimulation of renin release. This was accomplished using isolated perfused mouse kidneys with 3-dimensional electron microscopic analyses of renin-producing cells. Renin was found to be stored in a network of single granules and cavern-like structures, and dependent on the synthesis of glycosylated prorenin. Acute stimulation of renin release led to increased exocytosis in combination with intracellular fusion of vesicles to larger caverns and their subsequent emptying. Renin release from the kidneys of SCID-beige mice, which contain few but gigantic renin-storage vesicles, was no different from that of kidneys from wild-type mice. Thus, our findings suggest that renin is released by mechanisms similar to compound exocytosis. Kidney International (2013) 83, 233–241; doi:10.1038/ki.2012.392; published online 12 December 2012 KEYWORDS: afferent arteriole; angiotensin; renin–angiotensin system
Correspondence: Armin Kurtz, Physiologisches Institut der Universita¨t Regensburg, D-93053 Regensburg, Germany. E-mail: [email protected] Received 8 August 2012; revised 10 September 2012; accepted 13 September 2012; published online 12 December 2012 Kidney International (2013) 83, 233–241
The protease renin is the key enzyme regulating the activity of the renin–angiotensin–aldosterone system.1 It is mainly produced and secreted by specialized kidney cells located in the terminal parts of afferent arterioles.2,3 In these cells, renin is synthesized as a precursor protein prorenin, which is glycosylated in the Golgi apparatus and then transferred into storage vesicles.1,4–6 Within these storage vesicles, prorenin is proteolytically processed to renin.5 The release of renin from the cells is mainly controlled by two oppositely acting signaling pathways. The cyclic adenosine monophosphate (cAMP) signaling pathway stimulates renin release, whereas a calcium-related signaling pathway inhibits the release of renin.7–10 Inhibition of secretion by calcium is unusual, as calcium is considered to be essential for the induction or maintainance of secretory events, in particular of exocytosis.11 In fact, morphological signs of exocytosis, such as omega-shaped figures, have very rarely been reported in renin-producing cells,12,13 in spite of the numerous and prominent renin-containing vesicles, which often show an irregular shape at the ultrastructural level.14,15 Therefore, it has been hypothesized that the release of renin from storage vesicles may occur through intracellular solubilization of renin, which then is transported through the plasma membrane.16,17 Functional data such as a quantlike release of renin,18 increase of membrane capacitance19,20, and the disappearance of renin-storage vesicles upon stimulation of renin release,21 however, would support the idea of a controlled exocytosis of renin. Others have described membrane invaginations in the course of renin release.22–24 Signs of exocytosis of renin-containing vesicles such as omega-shaped membrane structures have been occasionally described for kidneys of animals, in which renin synthesis and secretion had been stimulated in vivo by adrenalectomy in combination with treatment with the loop diurectics.12 Notably, no signs of exocytosis have been reported for animals chronically treated with angiotensinconverting-enzyme (ACE) inhibitors,22 although the number of renin-storage vesicles clearly decreases during acute ACE-I treatment.22,25 In addition, periods of acute renal hypoperfusion lead to a decrease of the renin-storage vesicle number without morphological signs of exocytosis.22 Finally, beige mice lacking the Lyst protein produce only very few but huge renin-storage vesicles, without signs of 233
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RESULTS
Renin secretion rates of normal mouse kidneys perfused under standard conditions reached stable renin release rates after 15 min (Figure 1). After a control perfusion period of 5 min, kidneys were perfusion-fixed for electron microscopic analysis. Renin-producing cells in the juxtaglomerular areas contained numerous electron-dense vesicles that frequently showed an irregular circumference on 2D sections (Figure 2a). Up to 100 serial sections per cell were prepared to generate a 3D picture of renin-storing vesicles. These 3D reconstructions revealed that different forms of vesicles existed, ranging from single granules to huge interconnected caverns (Figure 2b and c). We analyzed a total of six cells from three kidneys, which all displayed a rather similar appearance. The distribution of single granules and caverns showed some variability even among renin-producing cells of the same kidneys under control conditions, suggesting that granules and cisternae are states of a dynamic equilibrium (Figure 2d). Common to all of these cells was that the vesicles covered around 36% of the extranuclear space of the cells (Table 1). The average volume of a vesicle was around 0.6 fl (Table 1). In six individual cells under control conditions, we reconstructed up to 50% of the cell surfaces. However, we did 234
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exocytosis.26 The plasma renin concentration reflecting secreted renin, is lower in these mice, however, in proportion with a decreased number of renin-expressing cells in the kidneys. Therefore, renin release from individual reninproducing cells may not be very different between wild-type and beige mice. Experiments with a more defined stimulation of renin release have been performed with incubated kidney slices, which were incubated with isoprenaline, calcium chelators, furosemide, cytochalasin B, or combinations of these compounds.12,13 Again, morphological signs of exocytosis were very rarely seen. More evident was the appearance of electron-lucent and/or empty vesicles, which were considered as indirect signs for exocytosis.12,13 Notably, isoproterenol, which is a powerful and immediately acting stimulator of renin release, did not induce morphological changes of renin-secreting cells.13 It was the combination of the calcium chelator ethylene glycol tetraacetic acid (EGTA) with cytochalasin B that produced a partial emptying of renin-storage vesicles within 20 min of incubation.13 However, it is not clear as to whether the combination of these nonphysiological compounds might have induced effects that do not reflect the physiological release mode of renin. In summary, the mode of renin release is still rather mysterious in several aspects. For a better understanding of this process, we were therefore interested to learn more about the morphology of renin-storage vesicles and their possible changes during controlled modulation of renin release. For this goal, we combined the model of the isolated perfused mouse kidney, which allows us to modulate renin release under quasi physiological conditions with three-dimensional (3D) electron microscopy of renin-producing cells in the kidney.
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Figure 1 | Perfusion protocol and renin secretion rates of isolated wild-type mouse kidneys. After a stabilization period of 15 min, the samples were taken for the determination of basal renin release (control period C). For determination of the normal juxtaglomerular cell structure, kidneys were fixed at the end of the control period (time point 1). After the control period, isoproterenol (ISO, 5 nmol/l) was added to the perfusate. Five minutes later, kidneys were fixed for electron microscopical (ELMI) analysis (time point 2). Further, during isoproterenol infusion, ethylene glycol tetraacetic acid (EGTA) (2.5 m) was added to the perfusate to lower the extracellular concentration of calcium. At 5 min after the start of EGTA infusion, kidneys were fixed for ELMI analysis (time point 3). Renin secretion data are means±s.e.m. of nine kidneys for the control period, six kidneys for the period during ISO infusion only, and three kidneys after the start of EGTA infusion. AngI, angiotensin I.
not observe membrane figures indicative for exocytosis, such as omega-shaped structures. We also did not observe electron-lucent or empty vesicles in these cells. In parallel, we also perfused and analyzed kidneys that expressed the ren-2, but not the ren-1d, gene. In the perfusate of these kidneys, very low enzymatic renin activity (5 ng AngI/hmlg) could be measured, which, however, was not increased by isoproterenol, nor by lowering of calcium. In juxtaglomerular cells of these kidneys, electron-dense interconnected vesicular structures could be seen as well (Figure 3a–c). The average volume of these vesicles in ren-2 kidneys was clearly smaller than those found in ren-1d/ren-2 kidneys (Table 1). Moreover, the total vesicular volume in ren-2 kidneys was less than 20% of those measured in ren-1d/ ren-2 kidneys (Table 1). We next analyzed cells from ren-1d/ren-2 kidneys in which renin secretion was prestimulated by isoproterenol (5 nmol/l) for 5 min, which led to a fivefold increase of renin secretion from the isolated kidneys (Figure 1). In none of the six cells analyzed did we find signs of exocytosis or structural alterations of the vesicular renin-storage system (not shown). During isoproterenol infusion (5 nmol/l), we then lowered the extracellular concentration of calcium (by the addition of EGTA 2.5 mmol/l) for another 5 min. This maneuver led to a 20-fold stimulation of renin release over basal values (Figure 1). Now, rearrangment of the vesicles became apparent on 2D sections (Figure 4a). Larger caverns due to Kidney International (2013) 83, 233–241
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Table 1 | Average vesicle volumes and total vesicular volumes of juxtaglomerular cells Mouse strain and kidney perfusion protocol
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Volume ratio (vesicles/extranuclear cell volume) (%)
ren-1d þ / þ (WT) control ren-2 þ / þ control ren-1d þ / þ (WT) (5 min ISO þ 5 min ISO þ EGTA) ren-1d þ / þ (WT) (15 min ISO þ EGTA) SCID-Beige control
0.6±0.05 0.25±0.09* 1.9±0.3*
35.7±3.2 6±1* 40.5±2.9
2.5±0.3*
36.4±3.5
4.4±0.2*
40±3.9
Abbreviations: EGTA, ethylene glycol tetraacetic acid; ISO, isoproterenol; SCID, severe combined immunodeficiency; WT, wild type. Data are means±s.e.m. of 6 cells each. *Po0.05 versus ren-1d wild-type control.
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Figure 2 | Analysis of a wild-type juxtaglomerular cell after control perfusion according to the protocol shown in Figure 1. (a) Transmission electron microscopy section of the juxtaglomerular cell (original magnification 3800) shows vesicles of different sizes and forms. (b–d) 3D reconstruction of the cell with individual renin vesicles in different colors and the nucleus in brown color. Vesicle structure ranged from single granules (b) to interconnected caverns (c). The intracellular arrangement of vesicles/caverns is shown in d.
fusion of smaller vesicles developed (Figure 4b and c), and the average volume of the storage vesicle increased from 0.6 to 1.9 fl (Table 1). The proportion of total vesicle volume over extranuclear space was not changed (Table 1). In addition, distinct exocytoses became visible, which did not show preferential localization to the endothelial or to the adventitial site. Such excytoses resulted from single granules, but also from extensions of larger cisternae (Figure 4d). Prolonged strong stimulation with isoproterenol in the presence of low extracellular calcium concentration for 15 min (Figure 5) led to exocytoses and to marked changes of vesicle appearance (Figure 6a). Apart from vesicles with normal appearance, numerous vesicles with lower electron density showed up. Electron-lucent and empty vesicles appeared, which were mostly fused to one vesicular complex (Figure 6b). Some vesicles appeared as emptied structures. As a consequence of the fusion of the vesicles, the average volume of the storage vesicles increased further to 2.5 fl, but the proportion of total vesicle volume over extranuclear space remained unchanged (Table 1). Kidney International (2013) 83, 233–241
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Figure 3 | Analysis of a juxtaglomerular cell of a ren-2-expressing mouse after control perfusion according to the protocol shown in Figure 1. (a) Transmission electron microscopy section of the juxtaglomerular cell (original magnification 3800) shows a few irregularly shaped electron-dense vesicles. (b, c) 3D reconstruction of the cell, with renin vesicles shown in dark gray. Vesicles appear to be interconnected, forming cavern-like structures (c).
The sum of the findings obtained so far would indicate that during stimulation of renin release intracellular vesicle fusion rather than vesicle fission occurs, suggesting that vesicle fission is not required for the stimulation of renin secretion. To test for this conclusion, we analyzed renin secretion from kidneys of severe combined immunodeficiency (SCID)-beige mice that carry a defective Lyst-like protein. The Lyst-like protein is relevant for vesicle fission.27 Basal renin release from beige kidneys was not different from that of wild-type kidneys (Figure 7). Transmission electron microscopy (TEM) analysis of renin cells of beige kidneys revealed a few, huge vesicular structures (Figure 8a). 3D analysis confirmed the existence of a few but huge caverns 235
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Figure 4 | Analysis of a wild-type juxtaglomerular cell after subsequent perfusion with isoproterenol and ethylene glycol tetraacetic acid (EGTA) according to the protocol shown in Figure 1. (a) Transmission electron microscopy section of the juxtaglomerular cell (original magnification 3800) shows numerous irregularly shaped vesicles. (b, c) 3D reconstruction of the cell with individual renin vesicles in different colors and the nucleus in brown color. Intracellular caverns developed, which were larger than single vesicles. Distinct caverns are indicated by different colors. (d) Distinct exocytoses became visible. These exocytoses resulted from single granules, but also from extensions of larger caverns.
and few additional single granules (Figure 8b–d). The average volume of the vesicles was clearly increased (Table 1). Notably, the proportion of total vesicle volume over extranuclear volume space was similar to that of normal mice (Table 1). Under basal perfusion conditions, we did not notice signs of exocytosis in six cells of three kidneys examined. Stimulation of renin secretion by isoproterenol and by lowering of the extracellular concentration calcium for 5 min each produced renin secretion rates that were not clearly different between beige mice and normal mice (Figure 7). Both analysis of TEM images (Figure 9a) and 3D analysis (Figure 9b and c) of these cells revealed the appearance of exocytoses but no obvious signs of a rearrangement of the storage structures. The fusion of vesicles with the plasma membrane or intracellular fusion of vesicles requires physical contact between these structures. As our data did not provide 236
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Figure 6 | Analysis of a wild-type juxtaglomerular cell after perfusion with isoproterenol and ethylene glycol tetraacetic acid according to the protocol shown in Figure 5. (a) Transmission electron microscopy section of the juxtaglomerular cell (original magnification 3800). According to their electron densities, four populations of storage vesicles could be distinguished, namely vesicles with normal density (1), vesicles in transition from normal to lower density (2), vesicles with low density (electron-lucent) (3), and emptied vesicles (4). Electron-lucent and emptied vesicles are interconnected (indicated by arrowheads). (b) 3D reconstruction of the cell with the nucleus shown in medium-gray color. Apart from vesicles with normal appearance (dark gray), numerous vesicles with lower electron density became visible. Electron-lucent and emptied vesicles fused to huge caverns. One of these interconnected vesicle networks is depicted in light-gray color. Kidney International (2013) 83, 233–241
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Figure 7 | Perfusion protocol and renin secretion rates of isolated severe combined immunodeficiency (SCID)-beige mouse kidneys. After a stabilization period of 15 min, the samples were taken for the determination of basal renin release (control period C). For the determination of the normal juxtaglomerular cell structure, kidneys were fixed at the end of the control period (time point 1). After the control period, isoproterenol (ISO, 5 nmol/l) was added to the perfusate. Then, during isoproterenol infusion, ethylene glycol tetraacetic acid (EGTA) (2.5 mmol/l) was added to the perfusate to lower the extracellular concentration of calcium. At 5 min after the start of EDTA infusion, kidneys were fixed for ELMI analysis (time point 2). For comparison, renin secretion rates of wild-type kidneys (taken from Figure 1) are also shown. Renin secretion data are means±s.e.m. of six kidneys for control period and three kidneys after control period. AngI, angiotensin I.
evidence for a directed movement of vesicles during the stimulation of renin release, we considered the possibility that the contact of the membranes may be controlled by the microfilament network.28–30 We therefore investigated the influence of compounds on renin secretion that are known to disrupt the microfilament network, such as cytochalasin D, which interrupts the actin–myosin microfilament network,31 or lantrunculin, which disrupts the F-actin network.32 However, neither compound added to the perfusate of isolated kidneys exerted an effect on basal renin secretion. In addition, there was no obvious effect of these compounds on the stimulation of renin secretion by isoproterenol (10 nmol/l), nor on the inhibitory effect exerted by angiotensin II (1 nmol/l) (Figure 10). DISCUSSION
Our data show that renin-storage vesicles do not show a uniform appearance such as typical secretory vesicles known from endocrine and exocrine cells.33,34 At the single-section electron microscopic level, renin-storage vesicles display irregular circumferences, which has already been observed by several investigators in the past.14,15,35 Our 3D reconstructions demonstrate that these irregularly shaped forms are 2D sections of different states of storage structures ranging from single vesicles to huge interconnected caverns. As the relative proportion of these different structures shows a significant variability between renin cells of otherwise Kidney International (2013) 83, 233–241
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Figure 8 | Analysis of a juxtaglomerular cell of a severe combined immunodeficiency (SCID)-beige mouse after control perfusion according to the protocol shown in Figure 7. (a) Transmission electron microscopy section of the juxtaglomerular cell (original magnification 3800) shows a few and huge irregularly shaped vesicles with inclusion bodies. (b–d) 3D reconstruction of the cell with individual renin vesicles in different colors and the nucleus in brown color. The form of the vesicles ranged from voluminous caverns (b) to flat and extended pancake-formed structures (c). The arrangement of the vesicles/caverns is shown in d.
untreated kidneys, we assume that the development of single vesicles and large cisternae might be a dynamic interchangeable and reversible process that involves fragmentation of caverns into vesicles and refusion of single vesicles into caverns. Our data confirm the previous notion that the development of big electron-dense granules in juxtaglomerular cells depends on the synthesis of the glycosylated prorenin.4,36 The nonglycosylated Ren-2 protein is constitutively released in its enzymatically inactive proform.37,38 Nonetheless, also ren-2 juxtaglomerular cells show some rudiments of electron-dense vesicular structures, probably reflecting the principal capability of the reninexpressing cells to develop such special structures. Our study also confirms previous reports on the relevance of the Lyst protein for the formation of smaller reninstorage vesicles.26 Apparently, the Lyst protein is essential for the segmentation of renin-storage cisternae into smaller vesicles. 237
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Figure 9 | Analysis of a juxtaglomerular cell of a severe combined immunodeficiency (SCID)-beige mouse after subsequent perfusion with isoporoterenol and ethylene glycol tetraacetic acid according to the protocol shown in Figure 7. (a) Transmission electron microscopy section of the juxtaglomerular cell (original magnification 3800) shows irregularly shaped vesicles. (b, c) 3D reconstruction of the cell with individual renin vesicles in different colors and the nucleus in brown color. There is no obvious rearrangement of the vesicles (b) relative to the nonstimulated state (Figure 8). Exoxytoses became apparent (c).
Our data now show that controlled stimulation of renin secretion by increasing the cAMP concentration and by lowering the calcium concentration leads to the appearance of morphological signs of exocytosis. A 20-fold stimulation of renin secretion was associated with an estimated number of five exocytotic events per cell, which were not seen under nonstimulated conditions. With a more moderate stimulation of renin secretion by isoproterenol alone, we did not see signs of exocytosis, which is also in accordance with data obtained from kidney slices.13 Although a number of five exocytoses per cells is small in comparison with other secretory cells, we think that the temporal coincidence between the stimulation of renin secretion and the appearance of exocytoses further supports the concept that exocytosis of storage vesicles is the relevant 238
Figure 10 | Renin secretion rates from wild-type mouse kidneys perfused with lantrunculin or cytochalasin D. After a stabilization period of 15 min, samples were taken for the determination of basal renin release (control period C). After the control period, either vehicle, lantrunculin, or cytochalasin D at concentrations of 3 mmol/l (period D1) or 10 mmol/l (period D2) were added to the perfusate. Renin secretion was stimulated by adding isoproterenol, 10 nmol/l (period ISO), or inhibited by adding angiotensin II, 1 nmol/l (period angiotensin II (Ang II)), to the perfusate. Renin secretion data are means±s.e.m. of three kidneys each.
pathway for the release of active renin.19–22 Given that the observed exocytoses account for the renin secretion during stimulation, one would extrapolate that, on average, among five cells, one might show one exocytosis to maintain basal renin secretion rates of the kidneys. It is not unlikely that such a low number of exocytoses under basal conditions would have escaped our notion. Such a low number of exocytoses would be in good agreement with calculations already made by Taugner et al.12 That profound changes of renin secretion can occur in spite of moderate numbers of exocytotic events likely results from the intracellular fusion of vesicles, which formed huge complex networks, that appeared to be emptied or contained electron-lucent material in states of stimulated secretion. One fusion pore with the plasma membrane thus allows the release of the content of several vesicles. Such an exocytotic release mode of stored material is termed compound exocytosis,39 which occurs in many cell types such as Kidney International (2013) 83, 233–241
D Steppan et al.: Renin release by compound exocytosis
pancreatic acinar cells, mast cells, eosinophils, and neutrophils.39–43 Compound exocytosis in combination with few extrusion pores in the plasma membrane likely explains the deep ‘plasma membrane invaginations’ that have been previously described in electron microscopic examinations of renin cells22–24 and which have already been speculated to reflect compound exocytosis. They become even more prominent during strong stimulation of renin release, which leads to an emptying of the cisternae. Apparently, vesicle fusion rather than vesicle fission occurs during the stimulation of renin secretion opposite to other endocrine cells. For example, in insulin-secreting b cells of the pancreas, stimulation of secretion causes an increased formation of single vesicles in the subplasmalemnal area.44–46 The conclusion on the relevance of intracellular vesicle fusion for renin secretion is also supported by the observation that renin secretion from kidneys that lack the Lyst protein, and thus show impaired formation of smaller vesicles, is similar to renin secretion from normal kidneys. This finding is in line with in vivo data indicating that plasma renin concentrations and the number of renin cells are reduced in proportion in beige mice,26 suggesting that renin secretion per cell is not very different between beige and wild-type mice. Our analysis further revealed that fusion of reninstorage vesicle with the plasma membrane is not restricted to smaller vesicles, but does also occur with large renincontaining cisternae as in beige mice. We did not obtain signs for a directed movement of vesicles in the direction of the plasma membrane during stimulation of secretion, as it occurs in other secretory cells.44,47 Instead, one could imagine that stimulation of renin secretion increases the fusogeneity of vesicle membranes once they get into contact with each other by random movement.48 The increase of fusogeneity of vesicle membranes could be induced by modifications of vesicle proteins, but also by modifications of the myofilament skeleton of the cells. As it had been reported that cytochalasin B favors exocytosis of renin from kidney slices, we investigated the effects of drugs disrupting actin filaments, such as cytochalasin D31 and latrunculin,32 on renin secretion from isolated perfused kidneys. However, we did not obtain evidence that any of the drugs interfered with renin secretion, suggesting that the actin filament has a minor role for the control of renin release. It cannot be excluded that the observed effect of cytochalasin B on renin release from kidney slices might result from side effects such as inhibition of glucose uptake,49 which might cause energy depletion in incubated kidney slices. It will be a task for future research to define the proteins relevant for fusion of renin-storage vesicles among themselves and with the plasma membrane. Obvious candidates for investigation would be members of the SNARE family.50,51 Our pilot experiments in this direction, however, indicate that the most common SNARE proteins found in endocrine cells are either lacking or are expressed at very low levels only in renin-secreting cells of the kidney. Kidney International (2013) 83, 233–241
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Therefore, future research should focus on unraveling the components of the exocytosis machinery in reninsecreting cells. MATERIALS AND METHODS Animals Experiments were normally conducted in 12- to 20-week-old male C57BL/6J mice (Jackson Laboratories). We also used male homozygous C57BL/6J-Lystbg-J/J (Beige) mice (Jackson Laboratories, Bar Harbor, Maine), which were clearly identifiable by their fur color. Homozygous Ren1d Cre/Cre (Ren-2) mice were generated by backcrossing of Ren1d þ /Cre- Ren-2 mice, which were kindly provided by Ariel Gomez, University of Virginia.52 For genotyping of the Ren1d Cre/Cre mice, tail biopsies were performed, and DNA was extracted and tested for the presence of wild-type and mutant genes using the following primers: Ren1d-sense, 50 -GAAGGAGAGC AAAAGGTAAGAG-30 ; Ren1d-antisense, 50 -GTAGTAGAAGGGGG AGTTGTG-30 ; and Cre, 50 -TTGGTGTACGGTCAGTAAATTGGAC30 . All animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health and were approved by the local ethics committee. Isolated perfused mouse kidney The isolated perfused mouse kidney model has been described in detail elsewhere.53 Briefly, the animals were anesthetized with an intraperitoneal injection of Ketamin (50 mg/kg body weight, Curamed, Karlsruhe, Germany) and Xylazin (80 mg/kg body weight, Ratiopharm, Ulm, Germany); the abdominal aorta was cannulated, and the right kidney was excised, placed in a thermostated moistening chamber, and perfused at constant pressure (90 mm Hg). Using an electronic feedback control, perfusion pressure could be changed and held constant in a pressure range between 40 and 140 mm Hg. Finally, the renal vein was cannulated and the venous effluent was collected for the determination of renin activity and venous blood flow. The basic perfusion medium consisted of a modified Krebs–Henseleit solution supplemented with 6 g/100 ml bovine serum albumin and with freshly washed human red blood cells (a 10% hematocrit). For the determination of renin secretion rates, three samples of the venous effluent were taken in intervals of 2 min during each experimental period. Renin activity in the venous effluent was determined by radioimmunoassay (Byk & DiaSorin Diagnostics, Seelze, Karlsruhe, Germany) as described elsewhere.53 Renin secretion rates were calculated as the product of the renin activity and the venous flow rate (ml/ming kidney weight). Renin secretion was modulated by adding defined concentrations of isoproterenol, EGTA, angiotensin II, latrunculin, or cytochalasin D (all from Sigma, Deisenhofen, Germany) to the perfusate. Fixation for transmission electron microscopy Kidneys were fixated with constant pressure (90 mm Hg) for 3 min by perfusion with phosphate-buffered saline (PBS) buffer containing 2% glutaraldehyde. The kidney was cut in half and stored at 4 1C in 2% glutaraldehyde/PBS until embedment for TEM. For any experimental condition, three kidneys were fixated. Embedment and transmission electron microscopy The kidney tissue was cut in 1-mm3-wide blocks and embedded in epoxyde resin (epoxy embedment kit, Fluka, Neu-Ulm, Germany) 239
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using an automatic microwave (Leica EM AMV, Leica, Germany). The embedded tissue was cut into 70-nm-thick serial slices using an ultramicrotome (EM UC7, Leica, Wetzlar, Germany), which were then placed on copper grids coated with pioloform. The serial slices were contrasted using a 4% uranyl acetate solution and a 0.5% lead citrate solution. For the acquisition of the images of a juxtaglomerular cell, a transmission electron microscope (Phillips CM12 TEM, Fei & Co, Eindhoven, Netherlands) with a LaB5 cathode and an acceleration voltage of 120 keV was used. The digitalization was carried out with a TEM-1000 slow-scan CCD camera and the program EM-Menu 4.0 (both from TVIPS-Tietz GmbH, Gauting, Germany). 3D tissue reconstruction We used a variation of the technique originally described by Sauter et al.54 The resulting serial images were converted to an image stack with constant picture size with the graphics software ImageJ. The image stack containing the sequence of B50–100 TEM images of a juxtaglomerular cell was then imported into the Amira 5.4.1 Visualization Software (Visage Imaging, Berlin, Germany). After correct scaling and alignment, various electron-dense structures of the cell were labeled and assigned to different defined materials. Finally, the surfaces were computed from the material data, resulting in a 3D model of the juxtaglomerular cell. For every experimental condition, two cells were reconstructed per kidney.
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14. 15.
16.
17.
18. 19.
20.
21. 22.
23.
24.
25.
DISCLOSURE
All the authors declared no competing interest. ACKNOWLEDGMENTS
The technical assistance provided by Marlies Hamann and Robert Go¨tz is gratefully acknowledged. We thank Frank Schweda for critical discussion. The study was financially supported by funds of the Deutsche Forschungsgemeinschaft (SFB 699).
26. 27.
28. 29. 30.
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