Structural analysis suggests that renin is released by compound exocytosis  Dominik Steppan, Anita Zügner, Reinhard Rachel, Armin Kurtz  Kidney International  Volume 83, Issue 2, Pages 233-241 (February 2013) DOI: 10.1038/ki.2012.392 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Perfusion protocol and renin secretion rates of isolated wild-type mouse kidneys. After a stabilization period of 15min, 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, 5nmol/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) (2.5m) was added to the perfusate to lower the extracellular concentration of calcium. At 5min 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. Kidney International 2013 83, 233-241DOI: (10.1038/ki.2012.392) Copyright © 2013 International Society of Nephrology Terms and Conditions

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. Kidney International 2013 83, 233-241DOI: (10.1038/ki.2012.392) Copyright © 2013 International Society of Nephrology Terms and Conditions

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). Kidney International 2013 83, 233-241DOI: (10.1038/ki.2012.392) Copyright © 2013 International Society of Nephrology Terms and Conditions

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. Kidney International 2013 83, 233-241DOI: (10.1038/ki.2012.392) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Perfusion protocol and renin secretion rates of isolated wild-type mouse kidneys. After a stabilization period of 15min, samples were taken for the determination of basal renin release (control period C). After the control period, isoproterenol (ISO, 5nmol/l) in combination with ethylene glycol tetraacetic acid (EGTA) (2.5mmol/l) was added to the perfusate. After 15min, kidneys were fixed for ELMI analysis (time point F). Renin secretion data are means±s.e.m. of three kidneys. AngI, angiotensin I. Kidney International 2013 83, 233-241DOI: (10.1038/ki.2012.392) Copyright © 2013 International Society of Nephrology Terms and Conditions

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, 233-241DOI: (10.1038/ki.2012.392) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 Perfusion protocol and renin secretion rates of isolated severe combined immunodeficiency (SCID)-beige mouse kidneys. After a stabilization period of 15min, 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, 5nmol/l) was added to the perfusate. Then, during isoproterenol infusion, ethylene glycol tetraacetic acid (EGTA) (2.5mmol/l) was added to the perfusate to lower the extracellular concentration of calcium. At 5min 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. Kidney International 2013 83, 233-241DOI: (10.1038/ki.2012.392) Copyright © 2013 International Society of Nephrology Terms and Conditions

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. Kidney International 2013 83, 233-241DOI: (10.1038/ki.2012.392) Copyright © 2013 International Society of Nephrology Terms and Conditions

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). Kidney International 2013 83, 233-241DOI: (10.1038/ki.2012.392) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 10 Renin secretion rates from wild-type mouse kidneys perfused with lantrunculin or cytochalasin D. After a stabilization period of 15min, 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 3μmol/l (period D1) or 10μmol/l (period D2) were added to the perfusate. Renin secretion was stimulated by adding isoproterenol, 10nmol/l (period ISO), or inhibited by adding angiotensin II, 1nmol/l (period angiotensin II (Ang II)), to the perfusate. Renin secretion data are means±s.e.m. of three kidneys each. Kidney International 2013 83, 233-241DOI: (10.1038/ki.2012.392) Copyright © 2013 International Society of Nephrology Terms and Conditions