Renin release from renal JG cells is essential for kidney development and the control of blood pressure. Although the hormonal and paracrine control of renin release has been extensively studied, the proteins involved and the biological mechanism mediating renin release have not been identified. Moreover, whether renin release from granules requires fusion with the plasma membrane has not been directly demonstrated. In several endocrine and secretory cells, specific SNARE proteins mediate granule exocytosis and peptide/hormone release (57
). However, the expression and role SNAREs in renin release have not been addressed. One of the reasons why SNAREs and fusion were not explored might be the paradoxical inhibitory effect of calcium on renin release that opposes the necessary role of calcium on SNARE complex zippering. Our data show for the first time that the SNARE machinery is expressed in JG cells and the VAMP isoform VAMP2 localizes to renin-containing granules and mediates cAMP-stimulated renin release. These are the first data identifying a trafficking protein involved in renin release. More importantly, the requirement of the SNARE protein, VAMP2, on renin release implies that the renin granule undergoes exocytosis.
We observed differential localization of VAMP2 and VAMP3 in JG cells. Most VAMP2 localized to renin-containing granules, whereas VAMP3 was excluded from renin granules and present in smaller vesicles and in a perinuclear pattern. Although our data indicate that VAMP3 is not involved in constitutive or regulated renin release, VAMP3 may play additional roles in JG cells. Similar to our results, in other exocrine cells VAMP3 was found to be mainly localized to organelles involved in the biosynthetic trafficking pathway rather than in controlling final steps of vesicle or exocytosis (27
To study the role of VAMP2 and -3 on renin release, we first used the clostridial tetanus toxin. Tetanus toxin recognizes and cleaves a sequence uniquely present in VAMP2 and -3 (44
). In intact (non-permeabilized) JG cells, tetanus toxin was efficiently internalized and cleaved ~50% of the total pool of VAMP2 and VAMP3. This effect was sufficient to block cAMP-stimulated renin release by 50–60%. It is possible that incomplete VAMP2 or VAMP3 inactivation explains the partial effect of tetanus toxin. However, higher concentrations (100 nm
) of the toxin did not further inhibit renin release (data not shown). Tetanus toxin inhibition of renin release did not affect processing of renin from its pro-form as total renin content in JG cell lysates did not change when compared with controls. The role of VAMP2 on exocytosis rather than renin processing is further supported by the inhibition of cAMP-stimulated exocytosis, as measured with FM1–43 membrane labeling.
To specifically address the role of these VAMPs, we knocked down VAMP2 and VAMP3 by shRNA. Gene silencing in primary cells is difficult; thus, we used adenoviral vectors with VAMP2 or VAMP3 shRNA sequences. Our data show that a 50–60% protein knockdown of VAMP2 inhibits cAMP-stimulated renin release by 50%, whereas silencing VAMP3 with similar efficacy had no effect. Although these data clearly support a role of VAMP2 in mediating renin release, we cannot rule out the involvement of other VAMPs. It is possible that VAMP7 or -8 (which are not cleaved by tetanus toxin) may be involved in mediating part (30–50%) of the stimulatory effect of cAMP on granule fusion with the plasma membrane. This would imply that there may be at least two pools of granules containing mature renin, perhaps with different SNARES. However, it is also possible that upon decrease or inactivation of VAMP2, other VAMP isoforms exert a compensatory role, as it occurs in other secretory cells (60
). Taken together our data demonstrate that VAMP2 but not VAMP3 mediates most of the cAMP-stimulated renin release, although the partial involvement of other isoforms cannot be ruled out and will require further investigation.
Base-line (non-stimulated) renin release was not affected by either tetanus toxin or by selective knockdown of either VAMP2 or VAMP3. These data indicate that VAMP2 participates in the regulated rather than the constitutive release of renin or in its maturation process. It is likely that different VAMPs mediate the constitutive and the regulated renin pathways as reported in other exocrine cells (22
). In addition, we found that inactivation or knockdown of VAMP2 and VAMP3 with tetanus toxin or silencing RNA did not affect the amount of total renin in JG cells (renin content). Thus, the inhibitory effect of silencing VAMP2 is not likely to be due to a decrease in the amount of mature renin in JG cells or defective granule maturation. Rather, our data point to VAMP2 being necessary for granule-to plasma membrane fusion (exocytosis per se
, the amount of renin released into the circulation represents a very small percentage of the total renin content (61
). Renin release from JG cells in isolated afferent arteriole is ~2–5% of the total renin content (15
). For our experiments, we have used an extensively characterized preparation of primary JG cells that is at least 80% pure and responds to physiological stimuli (9
). Although the percent of total renin released may seem small, our data are in excellent agreement with previous work studying JG cells in vivo
or after isolation (15
). This magnitude of response is similar to most endocrine cells, which release a small fraction of it granular contents upon stimulation (24
). We found that culturing JG cells for up to 72 h did not result in dedifferentiation, as renin release values under constitutive or stimulated conditions were comparable with those of cells cultured for 24 or 48 h. In addition, longer incubation times and viral transduction per se
did not affect renin release or renin content values.
At least two other SNARE proteins, a syntaxin and SNAP, are required to mediate granule exocytosis. The expression of these SNAREs has not been studied in JG cells. In other endocrine cells where exocytosis is stimulated by cAMP, VAMP2 pairs primarily with syntaxin-1, -3, and -4 and SNAP23. We found that syntaxin-1, -2 -3, and -4 and SNAP23 (but not SNAP25) are present in JG cells. In addition, we also found that other VAMP isoforms (VAMP4, -5, -7 and -8) as well as the accessory proteins Munc18 (Munc18a, -b, and -c) are expressed in JG cells (). Although the full fusogenic machinery seems to be present in JG cells, we did not find a differential pattern of enrichment for those SNAREs in JG cells. This is somehow predictable as it is likely that the different SNAREs are involved in different biological processes within the JG cells as well as kidney cell types. Furthermore, some of the isoforms that we found to be expressed in JG cells may be involved in renin granule maturation. Thus, it is essential to study the VAMP2-interacting SNARE proteins individually and their specific involvement on cAMP-stimulated renin granule exocytosis.
The molecular mechanisms by which cAMP increases renin release via VAMP2 are not known. Previous studies have shown that SNARE complex and exocytosis can be regulated directly by phosphorylation in other secretory cells (57
). It is possible that cAMP via PKA could be directly responsible for phosphorylation of VAMP2 binding partners such as SNAP23 (57
). In addition there are several proteins that modulate SNARE-mediated exocytosis such as snapin, tomosyn, and complexins (68
), which are targets for the cAMP-PKA pathways. Any of these may be present in JG cells and could subsequently enhance fusion of pre-docked dense core renin granules to the plasma membrane, but these have not been studied.
In most cells studied SNARE zippering and complex formation, which mediates the ultimately step in membrane fusion, requires calcium (22
). In addition, in most secretory cells studied, calcium is the triggering second messenger for exocytosis (22
). Opposite to most cells, in JG cells a decrease in intracellular calcium stimulates renin release, whereas high calcium inhibits it, a phenomenon known as the calcium paradox (7
). Although it might seem contradictory that SNAREs mediate renin release with an opposing effect of intracellular calcium on secretion, there are several potential explanations. One possible explanation is that a localized calcium spike or calcium microdomain occurs at the plasma membrane specifically near fusion sites without affecting intracellular calcium levels or stores (72
). In addition, some SNAREs are less sensitive to the requirement for calcium to induce exocytosis. For example, SNAP23 is less sensitive to changes in calcium concentration than SNAP25 (73
). Synaptotagmins, which are SNARE complex modulatory proteins, serve as calcium sensors (74
). Another possible explanation could be that the synaptotagmin isoforms mediating renin release in JG cells are calcium-independent as described earlier (74
). Thus, it is possible that the VAMP2-mediated renin release primarily involves a set of SNAREs that does not require an increase in calcium for zippering. However, this remains speculative and requires future investigation.
From all of the hypertensive patient population, less than 30% possess the expectedly low plasma renin activity (75
). The remaining percentage exhibits either normal or high plasma renin values, both of which are unexpected or “abnormal” as renin should be suppressed by higher blood pressure. The mechanisms that mediate these higher than normal renin levels in hypertension and whether they involve enhanced renin release and alterations in the proteins mediating renin secretion or signaling in JG cells have not been elucidated. Our findings identify for the first time that the SNARE protein VAMP2, but not VAMP3, mediates cAMP-stimulated renin release. Future studies focusing on the selective SNARE pairs from the syntaxin family and the mechanisms that modulate SNARE complex formation in JG cells would advance our understanding of renin exocytosis and may provide novel targets for inhibition of renin release.