RAS has been portrayed as an endocrine, paracrine, autocrine, and intracrine system (19-21) and thus it has been difficult to delineate the quantitative contributions of sys-temically delivered vs locally formed Ang peptides to the levels existing in any given tissue. Emerging evidence indicates that local formation is of major significance in the regulation of the Ang peptide levels in many organs and tissues (22,23). Various studies have demonstrated the importance of tissue RAS in the brain, heart, adrenal glands, vasculature, as well as in the kidney (1,24). Although every organ in the body has the elements of the RAS, the kidney is unique in having every component of the RAS with compartmentalization in the tubular and interstitial networks as well as intracellular accumulation. In addition, the kidneys, as well as the adrenal glands, have tissue concentrations of Ang II much greater than can be explained by the concentrations delivered by the arterial blood flow (25-28). There is substantial evidence that the major fraction of Ang II present in renal tissues is generated locally from the AGT delivered to the kidney and from the AGT locally produced by proximal tubule cells. Ang I delivered to the kidney can also be converted to Ang II (29,30). Renin secreted by the juxtaglomerular apparatus (JGA) cells and delivered to the renal interstitium and vascular compartment also provides a pathway for the local generation of Ang I. ACE is abundant in the rat kidney and has been located in the proximal and distal tubules, the collecting ducts and renal endothelial cells (31-33). Therefore, all of the components necessary to generate intrarenal Ang II are present along the nephron.
Although in the strictest sense renin is not a hormone, it can be considered as such because of its role in determining Ang I generation and because it is subject to tight control. Hence, the plasma renin concentration or activity is often used as a measure of the overall activity of the RAS. In most species, renin synthesized by JGA cells is the primary source of both circulating and intrarenal renin levels. However, some strains of mice also produce substantial amounts of renin in the submandibular and submaxillary glands as expression of the duplicated renin gene, Ren-2 (34,35).
The secreted active form of renin contains 339-343 amino acid residues after proteolytic removal of the 43-amino acid residue at the N-terminus (prorenin). Although circulating active renin is derived exclusively from the kidney, the kidneys and other tissues also secrete prorenin into the circulation and its concentration may exceed that of renin (36). Besides serving as the precursor for active renin, it has been suggested that circulating prorenin is taken up by some tissues through which it may contribute to the local synthesis of angiotensin peptides (37). In the heart under normal conditions, renin is not produced and its transcript is undetectable or extremely low (38,39). Nevertheless, transgenic rats expressing the Ren-2 renin gene exhibit high circulating prorenin levels in absence of the cardiac transgene owing to prorenin internalization into cardiomyocytes with the generation of angiotensins (40). These effects suggest that the uptake of circulating prorenin but not active renin may play an important role in cardiac hypertrophy.
Although there have been suggestions that renin itself or perhaps prorenin may directly elicit cellular effects, independent of the generation of Ang II, the only well-established role of renin is to act on AGT, a protein with a non-glycosylated weight of 52 kDa and synthesized primarily by the liver to form the decapeptide Ang I. Recent evidence indicates the existence of a renin receptor that may also initiate intracellular signaling to activate ERK1/ERK2 (41). In the heart and kidney, the recently described renin receptor (41-44) binds renin and prorenin leading to an increase in the catalytic efficiency of Ang I formation from AGT.
It should be recognized that JGA cells are not the only intrarenal structures in which renin has been localized. Kidneys from rats that are treated chronically with ACE inhibitors also exhibit renin immunoreactivity on the afferent arteriole extending well beyond the JGA loci up toward the interlobular artery, suggesting that ACE inhibition induces a recruitment of cells that in the basal state were not expressing the renin gene (45). Positive renin immunoreaction has also been observed in cells of glomeruli and of proximal and distal nephron segments (46-49). In addition, renin mRNA and protein expression have been also reported in proximal and distal nephron segments (47,49-53). Using immunoblotting, Rohrwasser et al. (53) found that renin was secreted by microdissected arcades of connecting tubule cells indicating that renin is probably secreted into the distal tubular fluid, leading to Ang I formation within the distal nephron lumen.
Ang I is rapidly converted into the major effector of this system, Ang II, by ACE, which is located on endothelial cells in many vascular beds and on membranes of various other cells including brush border membranes of proximal tubules (54,55). The localization of ACE within the kidney in various species has been well characterized. However, there are some important differences between humans and commonly used experimental animals (56). Indeed, Metzger et al. (56) reported that kidneys from normal human subjects predominantly expressed ACE in the brush border of proximal tubular segments, and very little ACE expression was observed on vascular endothelial cells. ACE was not detectable in the vasculature of the glomerular tuft, or even in the baso-lateral membranes of epithelial cells. In contrast, there was intense labeling on the endothelial cells of almost all the renal microvasculature of rats. However, kidneys from human subjects with non-neoplastic diseases manifested increased expression on vascular endothelial cells (56). Data indicating much lower endothelial expression in renal vascular endothelial cells in humans help explain the much lower Ang I to Ang II conversion rates that have been reported for human kidneys as compared to other species (57,58). Danser et al. (57) reported that less than 10% of arterially delivered Ang I is converted to Ang II, which is much lower than reported for dogs (59). The reduced ACE on renal vascular endothelial cells in humans implies that the influence of intrarenal Ang II formed from circulating precursors may have less significance than in experimental animals.
The recently described ACE2, which acts on Ang II to form Ang 1-7, is a membrane-associated and secreted enzyme expressed predominantly on endothelium, but highly restricted to heart, kidneys, and testis in humans (11). It has been suggested that Ang 1-7 acts on its own receptor, postulated to be the orphan Mas receptor (60,61). Recent studies demonstrated that genetic deletion of the G protein-coupled receptor encoded by the Mas protooncogene abolished the binding of Ang 1-7 to mouse renal cells (61). Ang 1-7 is thought to serve as an endogenous antagonist of the Ang II-induced actions mediated via ATj receptors (62). Thus, ACE2 could have substantial impact on the balance of Ang peptides found in the kidney by diverting the RAS cascade from Ang II to Ang 1-7. This helps explain the elevated Ang II levels in the ACE2 knockout mice (61). Collectrin, a novel homolog of ACE2, has been identified in mouse, rat and humans (63,64). Both ACE2 and collectrin have tissue-restricted expression in the kidney. Collectrin is localized on the luminal surface and in the cytoplasm of collecting duct cells and its mRNA is expressed in renal collecting ducts cells (64), whereas ACE2 is present throughout the endothelium and in proximal tubular epithelial cells. Collectrin is upregulated in the hypertrophic phase of the ablated kidney in 5/6 nephrectomized rat model (65). In contrast to ACE and ACE2, collectrin does not contain dipeptidyl carboxy-peptidase domains, and thus it may play a role in the hypertrophic phase through other pathways. Other peptides with reported biological activity formed as part of the Ang cascade include Ang 2-8 and Ang 3-8 as a consequence of action by aminopeptidases and other degradating enzymes (66). Although there is an increasing interest in the potential roles of these other Ang peptides, the majority of the evidence continues to support the established premise that most of the vascular and transport actions attributed to the RAS that lead to vascular constriction, enhanced sodium transport and hypertension are because of the actions of Ang II and also Ang III or Ang 2-8 acting primarily on ATj receptors (67-69). Nevertheless, Ang 1-7, Ang IV and Ang II-mediated activation of AT2 receptors may exert significant counteracting or protective actions partially buffering the ATi-mediated effects under certain circumstances (8,70-73).
Although most of the circulating AGT is produced and secreted by the liver, it is also produced by the kidneys. Intrarenal AGT mRNA and protein have been localized to proximal tubule cells indicating that the intratubular Ang II could be derived from locally formed and secreted AGT (27,74-76). Furthermore, AGT is regulated by an amplification mechanism such that AGT mRNA and protein are enhanced by Ang II (77). This effect helps to maintain or even increase further the production of Ang II in Ang II-dependent hypertension (74,75). The AGT produced in proximal tubule cells appears to be secreted directly into the tubular lumen in addition to producing its metabolites intracellularly and secreting them into the tubule lumen (27,51,53,78,79). Ding et al. (80) demonstrated in mice harboring the gene for human AGT fused to the kidney androgen-protein promoter that human AGT was localized primarily to proximal tubule cells. They found abundant human AGT in the urine but only slight traces in the systemic circulation. This finding suggests that most of the AGT formed in proximal tubule cells is destined for secretion into the lumen. Rohrwasser et al. (53) demonstrated luminal localization of AGT in proximal tubular cells in vivo and showed, in monolayer proximal tubule cell cultures, that most of the AGT was detected near the apical membrane. They also reported that AGT was detected at low nanomolar concentrations in urine from mice and human volunteers.
Proximal tubule AGT concentrations in anesthetized rats have been reported in the range of 300 nM, which greatly exceed the free Ang I and Ang II tubular fluid concentrations (81). Because of its molecular size, it seems unlikely that much of the plasma AGT filters across the glomerular membrane further supporting the concept that proximal tubule cells secrete AGT directly into the tubule (53,76,80,82). Kobori et al. (83) infused human AGT into hypertensive rats in order to determine whether AGT from the circulation could be detected in the urine. The human AGT was not detectable suggesting that most of the AGT in the urine is of renal origin (83). Formation of Ang I and II in the tubular lumen subsequent to AGT secretion may be possible because some renin is filtered and/or secreted from JGA cells. The identification of renin in distal nephron segments also indicates a possible pathway for Ang I generation from proximally delivered AGT. Intact AGT in urine reflects its presence throughout the nephron and, to the extent that renin and ACE are available along the nephron, substrate availability supports continued Ang I generation and Ang II conversion in distal segments (22,51,53,80).
Once Ang I is formed, conversion readily occurs because there are abundant amounts of ACE associated with the proximal tubule brush border. Casarini et al. (32) found that ACE activity is present in tubular fluid throughout the nephron except in the late distal tubule, being higher at the initial portion of the proximal tubule but then decreasing to the distal nephron and increasing again in the urine. This evidence suggests proximal ACE secretion, degradation, and/or reabsorption associated with secretion in the collecting duct. Therefore, intratubular Ang II formation may occur not only in the proximal tubule but also beyond the connecting tubule. Thus, renal tissue ACE activity is critical to maintain the steady-state Ang II levels in the kidney. Indeed, Modrall et al. (84) demonstrated that tissue-ACE (tfsACE-/-) knockout mice exhibit 80% lower intrarenal Ang II levels compared to wild-type mice (84). In addition to the marked reduction of intrarenal Ang II levels, this tfsACE-/- mouse model showed significant depletion of its immediate precursor Ang I in renal tissue, which supports the concept that Ang II exerts a positive feedback loop on proximal AGT (74,75,77). However, at present there are no data indicating how much of the Ang peptides are formed intracellularly and secreted and how much are formed in the tubule lumen from secreted substrate.
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