The Increasing Complexity of the Intratubular Renin-Angiotensin System
Authors: L. Gabriel Navar, Ryousuke Satou, Romer A. Gonzalez-Villalobos
Journal: Journal of the American Society of Nephrology (2012)

The presence of receptors for angiotensin II (AII) on the luminal membranes of various nephron segments has been well established for many decades.1,2 Originally their function remained unclear because tubular fluid AII concentrations were thought to be quite low due to the presence of various degrading enzymes on the brush border of proximal tubular cells. However, a series of reports in the 1990s demonstrated that the proximal tubular concentrations of AI and AII are in the nanomolar range and much higher than can be explained by tubular fluid reabsorption or equilibration with the circulating levels.3,4 These findings led to a paradigm shift in our concepts regarding the role of luminal AII receptors in various nephron segments, and it is now well accepted that intraluminal AII and other angiotensin peptides exert various actions on transport systems in essentially all nephron segments predominantly through activation of AT1 receptors.5–10 In further studies, proximal tubular fluid samples were incubated with excess renin to determine substrate availability. The resultant AI concentrations demonstrated that the proximal tubular fluid angiotensinogen (AGT) concentrations are also very high11 and greater than the circulating concentrations, indicating that it is unlikely the tubular AGT concentrations are derived from filtered AGT, in particular considering the limited permeability of the AGT because of its large size.11 Using in situ perfusion of proximal tubules with artificial tubular fluid with the delivery of filtrate blocked, Braam et al.4 collected the tubular fluid from downstream segments and found these also had elevated AII concentrations in the nanomolar range, supporting tubular secretion of AII or its precursors. Studies on isolated perfused tubules from S2 segments indicated that AII is produced intracellularly and secreted preformed into the tubular lumen, supporting the presence of intact AGT in isolated proximal tubule segments.12 These findings, along with clear evidence for the presence of AGT mRNA and protein in proximal tubular cells,13–15 provided the foundation for the concept that the intratubular AII concentrations are derived primarily from locally synthesized substrate. The local production of AGT in the proximal tubule was supported by Terada et al.,14 who demonstrated the presence of a large signal for AGT mRNA in microdissected proximal convoluted and straight tubules. In vitro studies have also consistently demonstrated mRNA encoding AGT in proximal tubular cell lines extracted from proximal tubule segments.15,16 However, Pohl et al.17 recently reported predominant localization of mRNA encoding AGT to S3 segments. The findings of kidney tubular mRNA encoding AGT notwithstanding, the kidney's ability to produce AGT is dwarfed by that of the liver, the organ primarily responsible for the maintenance of circulating AGT concentrations. In addition, it is well recognized that filtered AGT can be taken up by proximal tubule scavenger receptors such as megalin and cubilin,17,18 or by a putative-specific AGT receptor on proximal tubular epithelial cells.19 Thus, various studies demonstrated that AGT is internalized by these transporters, which is consistent with observations that megalin binds and internalizes AII.18 The important question of the quantitative contribution of either source to total renal AGT content remained unresolved because of the lack of definitive tools needed to provide the answer. The paper by Matsusaka et al.20 in this issue of JASN provides critical data obtained in mice that had either liver AGT or the kidney AGT mRNA expression deleted genetically. Their results show that kidney-specific AGT-null mice have kidney AGT protein and AII levels similar to those in control mice, whereas the liver-specific AGT-null mice have markedly lower or undetectable levels of AGT in the proximal tubules and kidney AII. Further studies confirmed that AGT uptake by proximal tubular cells is mediated by megalin and that disruption of the filtration barrier markedly increases tubular and urinary AGT protein. The results thus indicate that, in normal nonhypertensive mice, most of the AGT protein present in proximal tubules is of liver origin. This finding is not unexpected, as the kidney levels of AGT protein in normal rats and mice are rather low, especially in comparison to the enhanced levels that develop in response to chronic AII infusions.21–23 In addition, AGT synthesis and secretion is a constitutive process, so cells expressing mRNA encoding AGT probably do not store the protein, but secrete it into the tubular fluid. To the extent that the proximal tubule AII concentrations in mice are as high as have been found in rat proximal tubular fluid,24 the results presented by Matsusaka et al. pose the interesting dilemma regarding the cellular pathways by which the increased intratubular AII levels are derived from the AGT taken up from the circulation by the proximal tubular cells. AII may be formed intracellularly from AGT reabsorbed from either the renal interstitium or tubular fluid, or the internalized AGT may be resecreted into the tubular lumen where peptide generation takes place. Thus, a major experimental and conceptual challenge that emerges from these findings is to understand the cellular and molecular trafficking that occurs to accomplish these formidable processes. Because megalin is a scavenger receptor, its prevailing function has been generally considered to be the uptake of peptides and proteins that cross the glomerular filtration barrier and targeting them to degradation by the lysosomal pathway.25 However, new advances indicate that megalin may also act as a signaling molecule.26,27 Thus, the internalization of AGT may result in intracellular formation of AI and AII and/or signaling. These intracellularly generated angiotensins may be secreted into proximal tubular fluid. Although some of the internalized AGT may be resecreted into the proximal tubular fluid, an alternative pathway is that a part of the AGT-megalin complex is released by the megalin ectodomain shedding, leading to increases in AGT and AII concentrations in proximal tubular fluid. A critical issue not addressed by Matsusaka et al. is what the quantitative and functional contribution of either source of AGT is during hypertensive states or renal diseases.28 Studies in chronic AII-infused animals showed increased AGT protein accumulation in all segments of the proximal tubule.21,23,29 These results led to the hypothesis of a robust and independent tubular renin-angiotensin system where augmentation of proximal tubular AGT leads to increased intratubular secretion of AGT and greater intratubular formation of AI and AII to act on luminal AT1 receptors and stimulate tubular transport.24 Although Matsusaka et al. confirm that AGT in the urine is derived, in large part, from the mRNA encoding AGT present in latter segments of the proximal tubules, these levels are extremely low and can be markedly upregulated to yield many fold greater increases in urinary AGT concentrations as found in AII-infused animals.21,24,30 Urinary AII levels are markedly augmented when there is a stimulus for AGT overexpression in renal proximal tubules, demonstrating that AGT produced in proximal tubules contributes to intrarenal AII elevation and increased urinary AGT excretion.31 Thus, the study by Matsusaka et al. does not address contributions of intrarenal mRNA encoding AGT and locally synthesized AGT to tubular AII in AII-dependent hypertension or in various kidney diseases.28 The augmentation of proximal tubule AGT protein during hypertensive states cannot be attributed to megalin uptake, because it occurs despite substantial megalin downregulation by activated Extracellular signal regulated kinase1/2 and, in many instances, in advance of overt proteinuria.32 Furthermore, deoxycorticosterone-salt rats have proteinuria but do not have increased urinary AGT.33 Thus, the data support an important role of proximal tubule AGT, generated locally in hypertension. In this setting, it is possible that mRNA encoding AGT extends beyond the S3 segment to the convoluted segments as has been shown in vitro. Furthermore, these in vitro experiments show that AII and other factors including proinflammatory cytokines such as IL-6 and IFN-γ, glucocorticoids, reactive oxygen species, and glucose synergistically or independently stimulate the expression of mRNA encoding AGT mainly through NF-κB and JAK-STAT pathways in cultured proximal tubular cells.34,35 In summary, the report by Matsusaka et al. makes an important contribution to our understanding of the ever increasing complexity of the intrarenal renin-angiotensin system by demonstrating that, in nonstimulated states, liver AGT and not kidney AGT, is mainly responsible for kidney AII and proximal tubule AGT. However, the study does not address the influence of augmented mRNA encoding AGT in the kidneys that occurs in AII-dependent hypertension, diabetes, and certain other renal diseases.24,28 Disclosures None. We thank Debbie Olavarrieta for preparation of the manuscript. The authors received support from National Heart, Lung, and Blood Institute Grants HL-18426 and HL-26371, the Institutional Award Program of the National Center for Research Resources, Centers of Biomedical Research Excellence Grant P20 RR-017659, and National Institute of Diabetes and Digestive and Kidney Diseases Grant R00DK083455).