Delivery of B Cell Receptor–internalized Antigen to Endosomes and Class II Vesicles

B cell receptor (BCR)-mediated antigen processing is a mechanism that allows class II–restricted presentation of specific antigen by B cells at relatively low antigen concentrations. Although BCR-mediated antigen processing and class II peptide loading may occur within one or more endocytic compartments, the functions of these compartments and their relationships to endosomes and lysosomes remain uncertain. In murine B cells, at least one population of class II– containing endocytic vesicles (i.e., CIIV) has been identified and demonstrated to be distinct both physically and functionally from endosomes and lysosomes. We now demonstrate the delivery of BCR-internalized antigen to CIIV within the time frame during which BCR-mediated antigen processing and formation of peptide–class II complexes occurs. Only a fraction of the BCR-internalized antigen was delivered to CIIV, with the majority of internalized antigen being delivered to lysosomes that are largely class II negative. The extensive colocalization of BCR-internalized antigen and newly synthesized class II molecules in CIIV suggests that CIIV may represent a specialized subcellular compartment for BCR-mediated antigen processing. Additionally, we have identified a putative CIIV-marker protein, immunologically related to the Igα subunit of the BCR, which further illustrates the unique nature of these endocytic vesicles.The recognition of MHC class II–restricted antigens by antigen-specific T cells requires the proteolytic processing of protein antigens to immunogenic peptides by class II–positive antigen-presenting cells (1, 2). The first step in antigen processing by B cells involves B cell receptor (BCR)¹–mediated internalization of antigen (3–5). BCR-internalized antigen is then proteolytically processed and the resultant peptides preferentially loaded onto newly synthesized class II molecules (6–8) from which the class II– associated invariant chain has been removed by the concerted action of acid proteases and the protein HLA-DM/ H-2M (9). The resultant peptide–class II complexes are then transported to the surface of the B cell.The intracellular compartments where antigen processing occurs have only recently been characterized and there is considerable variation in the intracellular localization of class II molecules among different cell types. Many cells, such as human lymphoblasts and macrophages, sequester much of their class II in lysosomes or lysosome-like structures referred to as the MHC class II–enriched compartment (MIIC; reference 10). Although delivery of BCR-internalized antigen to MIIC has been demonstrated (11), the fate of the antigen delivered to these structures (i.e., complete degradation versus processing and binding to class II molecules) remains unknown.In other professional antigen-presenting cells such as many murine B cell lines, there is little accumulation of class II in lysosomes under normal conditions (12–14). Instead, class II is found in endosomes and endosome-related structures, at least one population of which (class II vesicles [CIIV]) can be purified and physically separated from conventional endocytic and secretory organelles by cell fractionation techniques (14).Although many or all endocytic, class II–containing vesicle populations may host the loading of peptides onto class II molecules, there may be important qualitative differences regarding the subcellular compartments where antigenic peptides are generated and efficiently loaded onto class II molecules. Specifically, although BCR-mediated antigen presentation appears to involve binding of peptide to newly synthesized class II molecules (6–8), presentation of fluid phase proteins by B cells appears to be able to occur via both newly synthesized and recycling class II molecules (7, 8, 15, 16), possibly reflecting differences in the intracellular sites of peptide generation and class II loading.Additionally, not all receptors are equivalent at mediating antigen processing and presentation. In murine B cells, antigen internalized via the transferrin receptor (while presented more efficiently than soluble antigen) is presented 10–100 times less efficiently than the same antigen internalized via the BCR (17). This result may reflect the fact that the transferrin receptor has far more restricted access to intracellular class II compartments in B cells than does the BCR (11). Even more dramatic is the demonstration that a single amino acid substitution in the transmembrane region of the human IgM BCR (huBCR) can completely abolish the ability of this receptor to mediate efficient antigen processing and presentation without affecting BCR-mediated antigen endocytosis and bulk antigen degradation (18, 19). Thus, antigen uptake and degradation is necessary, but not sufficient, for antigen processing and presentation.Thus, it has become important to determine the intracellular compartments to which physiologically important receptors (e.g., the BCR) deliver antigens. In this paper, we demonstrate that, within the time frame during which the intracellular events of BCR-mediated antigen processing are known to occur, BCR molecules and BCR-internalized antigen have access not only to predominantly class II– negative endosome and lysosomes, but also to a novel population of endocytic vesicles that are highly enriched in newly synthesized class II molecules (i.e., CIIV). Moreover, CIIV contain a putative marker protein, immunologically related to the Igα subunit of the BCR, further illustrating the distinct nature of these endocytic vesicles.     

Murine Transporter Associated with Antigen Presentation (TAP) Preferences Influence Class I–restricted T Cell Responses

The transporter associated with antigen presentation (TAP) complex shuttles cytosolic peptides into the exocytic compartment for association with nascent major histocompatibility complex class I molecules. Biochemical studies of murine and human TAP have established that substrate length and COOH-terminal residue identity are strong determinants of transport efficiency. However, the existence of these specificities in the intact cell and their influences on T cell responses have not been demonstrated. We have devised a method for studying TAP- mediated transport in intact cells, using T cell activation as a readout. The approach makes use of a panel of recombinant vaccinia viruses expressing peptides containing the K^d-restricted nonamer influenza nucleoprotein residues 147–155. The COOH terminus of each construct was appended with a dipeptide composed of an internal threonine residue followed by a varying amino acid. Synthetic peptide versions of these 11-mers exhibit vastly different transport capabilities in streptolysin O–permeabilized cells, in accordance with the predicted influence of the COOH-terminal residues. Presentation of the endogenously expressed version of each construct requires TAP-mediated transport and cooexpression with a vac-encoded exocytic COOH-terminal dipeptidase, angiotensin converting enzyme, to allow liberation of the minimal epitope. Recognition by epitope-specific CTLs therefore signifies TAP-mediated transport of a complete 11-mer within the target cell. Under normal assay conditions no influences of the COOH-terminal residue were revealed. However, when T cell recognition was limited, either by blocking CD8 coreceptor interactions or by decreasing the amount of transport substrate synthesized, significant COOH-terminal effects were revealed. Under such conditions, those peptides that transported poorly in biochemical assays were less efficiently presented. Therefore, TAP specificity operates in the intact cell, appears to reflect previously defined rules with regard to the influence of the COOH-terminal residue, and can strongly influence T cell responses.CD8⁺ CTLs recognize short (8–10-amino acid) peptide portions of antigen (epitopes) complexed with major histocompatibility class I molecules (1–3). The initial processing of most antigens destined for recognition by class I–restricted CTLs occurs in the cytosol. The resultant fragments are then conveyed to the exocytic compartment by the transporter associated with antigen presentation (TAP)¹ heterodimer where, perhaps after further processing, they become available for binding to nascent class I. Because of this function, the TAP complex, a member of the ABC family of transporters (4, 5), is critical for presentation of the vast majority of class I–restricted epitopes, as well as for the surface expression of class I molecules themselves (6).Numerous potential epitopes are contained within an antigenic protein, but very few trigger CTL responses. Although much of this selectivity can be attributed to stringent haplotype-specific class I binding requirements (7), other factors determining epitope immunogenicity include the availability of appropriate T cell specificities and the ability of the proteolytic machinery to excise the epitope without destroying it too rapidly (8, 9). As understanding of the critical role of TAP in class I–restricted antigen presentation has grown, it has been intriguing to speculate that TAP substrate specificity may also have a significant hand in determining which epitopes are available for T cell recognition.It was initially observed that polymorphism at the rat cim locus, the TAP homologue in that species, could be correlated with variability in the array of class I–associated peptides (10). This finding was of particular significance because it implied an influence of transport specificity in a relatively unmanipulated system. Although the cim effect could be shown to influence T cell responses in the rat, it is important to note that the cim locus exhibits far greater variability than does the TAP locus in either mice or humans (3). This approach, applied to a study of class I–associated peptides in mice and humans, has failed to identify a similar influence of allelic variation on TAP specificity (11, 12). However, in vitro biochemical assays, using either streptolysin O–permeabilized cells or isolated microsomes, have provided evidence for substrate preferences by all TAP alleles in both species (13–18). By these means, a size optimum of 8–12 residues has been established, and it has been found that mouse TAPs prefer peptides with hydrophobic COOH-terminal residues; human TAPs transport peptides with both hydrophobic and acidic COOH termini (19, 20). These preferences are consistent with MHC binding capabilities and this, along with the genetic linkage between TAP and MHC (3), has given rise to the speculation that the two have coevolved to enhance the efficiency of the class I–restricted response. To date, these biochemical assays provide the only evidence for murine or human TAP selectivity; as yet it is undemonstrated whether the measured preferences have significance for T cell recognition.Some evidence suggests that murine TAP specificity may not play a large role in shaping T cell responses. It is clear that substrates showing little or no transport capability in in vitro transport assays can be presented to T cells and serve as potent immunogens in vivo. Shepherd et al. previously reported that the minimal epitope influenza nucleoprotein residues 147–155 (NP_147–155) is not detectably transported into isolated microsomes, with a 50% inhibitory concentration (IC₅₀) value of >50 μM (14). Although these authors suggested that a longer fragment containing the epitope may be transported and further processed in the exocytic compartment, a minigene expressing only the minimal epitope is a more efficient immunogen than full-length NP (21, 22). Furthermore, it has been reported that cells expressing murine TAP and the human HLA-A3 gene product can efficiently produce ligands for this class I molecule despite its preference for positively charged COOH termini (23), which are likely to be generated before transport (24). Given the high sensitivity of T cells, it is possible that TAP selectivity may not significantly impact their responses under physiological conditions. Additionally, as with in vitro nuclear transport assays (25), streptolysin O and microsome isolation may lead to a loss of molecules that influence transport in vivo. It is possible that such molecules physically interact with the TAP complex, processed fragments, or both, to influence transport specificity and/or rates. Therefore, we wished to test whether biochemically established transport capability was in fact correlated with TAP selectivity within intact cells and, if so, begin to elucidate conditions of antigen expression and CTL sensitivity under which selectivity might influence an immune response.In this study, we describe a unique approach to measuring the influence of TAP specificity within live, intact cells, using the physiologically significant readout of CTL recognition. A previous study demonstrated that the K^d-restricted influenza nucleoprotein epitope, amino acids 147–155, cannot be processed and presented when appended by two COOH-terminal residues, threonine and glycine, due to the unavailability of appropriate proteolytic activity (21). Presentation of the epitope can take place when the fragment is coexpressed with the dipeptidyl carboxypeptidase angiotensin converting enzyme (ACE; references 26 and 27). As rescue is intracellular and TAP-dependent, and this enzyme is active only in the exocytic compartment, and ACE-mediated processing of the 11-mer is a reflection of TAP-mediated transport within intact target cells (see Fig. ​Fig.11 for a schematic of this process). We have used this system to examine class I–restricted presentation of an array of substrates that differ significantly in their in vitro transport capabilities. Open in a separate windowFigure 1Schematic diagram of transport-dependent ACE-mediated processing in the exocytic compartment.     

Arming Cytokine-induced Killer Cells With Chimeric Antigen Receptors: CD28 Outperforms Combined CD28–OX40 “Super-stimulation”

Cytokine-induced killer (CIK) cells raised interest for use in cellular antitumor therapy due to their capability to recognize and destroy autologous tumor cells in a HLA-independent fashion. The antitumor attack of CIK cells, predominantly consisting of terminally differentiated CD8⁺CD56⁺ cells, can be improved by redirecting by a chimeric antigen receptor (CAR) that recognizes the tumor cell and triggers CIK cell activation. The requirements for CIK cell activation were, however, so far less explored and are likely to be different from those of “younger” T cells. We revealed that CD28 and OX40 CARs produced higher interferon- secretion as compared with the first-generation ζ-CAR; CD28-ζ and the third-generation CD28-ζ–OX40 CAR, however, performed similar in modulating most CIK cell effector functions. Compared with the CD28-ζ CAR, however, the CD28-ζ–OX40 CAR accelerated terminal maturation of CD56⁺ CIK cells producing high frequencies in activation-induced cell death (AICD) and reduced antitumor efficiency in vivo. Consequently, CD28-ζ CAR CIK cells of CD56⁻ phenotype were superior in redirected tumor cell elimination. CAR-mediated CIK cell activation also increased antigen-independent target cell lysis; the CD28-ζ CAR was more efficient than the CD28-ζ–OX40 CAR. Translated into therapeutic strategies, CAR-redirected CIK cells benefit from CD28 costimulation; “super-costimulation” by the CD28-ζ–OX40 CAR, however, performed less in antitumor efficacy due to increased AICD.     

Fusion of HCV Nonstructural Antigen to MHC Class II–associated Invariant Chain Enhances T-cell Responses Induced by Vectored Vaccines in Nonhuman Primates

Despite viral vectors being potent inducers of antigen-specific T cells, strategies to further improve their immunogenicity are actively pursued. Of the numerous approaches investigated, fusion of the encoded antigen to major histocompatibility complex class II–associated invariant chain (Ii) has been reported to enhance CD8⁺ T-cell responses. We have previously shown that adenovirus vaccine encoding nonstructural (NS) hepatitis C virus (HCV) proteins induces potent T-cell responses in humans. However, even higher T-cell responses might be required to achieve efficacy against different HCV genotypes or therapeutic effect in chronically infected HCV patients. In this study, we assessed fusion of the HCV NS antigen to murine and human Ii expressed by the chimpanzee adenovirus vector ChAd3 or recombinant modified vaccinia Ankara in mice and nonhuman primates (NHPs). A dramatic increase was observed in outbred mice in which vaccination with ChAd3 expressing the fusion antigen resulted in a 10-fold increase in interferon-γ⁺ CD8⁺ T cells. In NHPs, CD8⁺ T-cell responses were enhanced and accelerated with vectors encoding the Ii-fused antigen. These data show for the first time that the enhancement induced by vector vaccines encoding li-fused antigen was not species specific and can be translated from mice to NHPs, opening the way for testing in humans.     

Recognition of Human Histocompatibility Leukocyte Antigen (HLA)-E Complexed with HLA Class I Signal Sequence–derived Peptides by CD94/NKG2 Confers Protection from Natural Killer Cell–mediated Lysis

Human histocompatibility leukocyte antigen (HLA)-E is a nonclassical HLA class I molecule, the gene for which is transcribed in most tissues. It has recently been reported that this molecule binds peptides derived from the signal sequence of HLA class I proteins; however, no function for HLA-E has yet been described. We show that natural killer (NK) cells can recognize target cells expressing HLA-E molecules on the cell surface and this interaction results in inhibition of the lytic process. Furthermore, HLA-E recognition is mediated primarily through the CD94/NKG2-A heterodimer, as CD94-specific, but not killer cell inhibitory receptor (KIR)–specific mAbs block HLA-E–mediated protection of target cells. Cell surface HLA-E could be increased by incubation with synthetic peptides corresponding to residues 3–11 from the signal sequences of a number of HLA class I molecules; however, only peptides which contained a Met at position 2 were capable of conferring resistance to NK-mediated lysis, whereas those having Thr at position 2 had no effect. Interestingly, HLA class I molecules previously correlated with CD94/NKG2 recognition all have Met at residue 4 of the signal sequence (position 2 of the HLA-E binding peptide), whereas those which have been reported not to interact with CD94/NKG2 have Thr at this position. Thus, these data show a function for HLA-E and suggest an alternative explanation for the apparent broad reactivity of CD94/NKG2 with HLA class I molecules; that CD94/NKG2 interacts with HLA-E complexed with signal sequence peptides derived from “protective” HLA class I alleles rather than directly interacting with classical HLA class I proteins.In humans, there are three classical class I MHC molecules (HLA-A, -B, and -C). These molecules consist of a peptide bound to a transmembrane glycoprotein subunit, the heavy chain, in association with a soluble light chain, β₂-microglobulin (1). Nonclassical MHC class I molecules show homology to classical class I molecules but generally have limited polymorphism, low cell surface expression, and more restricted tissue distribution (2). The function of these nonclassical class I molecules remains unclear, but some of them may have more specialized antigen presentation activities, e.g., mouse H2-M3 presents N-formylated peptides (3). In humans, the nonclassical HLA-G molecule binds a wide range of peptides derived from cellular proteins and has been suggested to play an important role in the maintenance of maternal tolerance to the fetus by interacting with inhibitory receptors on NK cells (4). HLA-E is another nonclassical class I molecule and, like classical MHC class I loci, the HLA-E gene is highly transcribed in many tissues (5–7). Mouse cell lines transfected with HLA-E and human β₂-microglobulin generally exhibit low levels of cell surface expression, which has been attributed to a lack of appropriate endogenous peptides (8). Recent in vitro studies have shown that HLA-E can bind peptides derived from the signal sequences of certain HLA class I molecules and that the primary peptide anchor residues are at positions 2 and 9 (9).NK cells are one of the three lineages of lymphocytes and are thought to control viral infections and tumor development (10). They are capable of killing MHC class I negative target cells without prior sensitization. Furthermore, the expression of class I molecules on target cells renders them resistant to lysis by most NK cells (11). Inhibitory receptors expressed by human NK cells belong to either the immunoglobulin (killer cell inhibitory receptor; KIR) or the C-type lectin superfamily. The best characterized members of the KIR family contain two (p58 molecules: CD158) or three (p70 molecules: NKB1) immunoglobulin-like domains and different receptors of this family bind defined groups of classical HLA class I molecules (12, 13). The second family of receptors is composed of two subunits: CD94 paired with one member of the NKG2 family of proteins. The CD94–NKG2-A heterodimer transmits signals that lead to the inhibition of the lytic process. Numerous studies suggest that this heterodimer recognizes a broad panel of classical HLA class I molecules (14–19).Recognition of nonclassical HLA class I molecules by NK cells has been recently described. Specifically, numerous reports suggest that HLA-G can confer protection from lysis to otherwise sensitive target cells, although the identity of the receptors involved in this interaction remains controversial (20–24). In this study we examined whether another nonclassical class I molecule, HLA-E, can be recognized by NK cells. Our results show that NK cells can interact with HLA-E complexed with specific peptides on target cells and that this recognition is mediated, at least partially, if not solely, by CD94–NKG2.     

Cancer Antigen 125 as a Biomarker in Peritoneal Dialysis: Mesothelial Cell Health or Death?

The concentration or appearance rate of cancer antigen 125 (CA125) in peritoneal dialysis (PD) effluent has been used for many years as a biomarker for mesothelial cell mass in patients on PD. However, this marker has limitations, and emerging evidence has raised doubts as to its significance. This review explores our current understanding of CA125, its prominent role in studies of “biocompatible” PD solutions, and the ongoing uncertainty concerning its interpretation as a measure of mesothelial cell health.Key words: Cancer antigen 125, CA125, mesothelial cell mass, biocompatibility, biomarkers, peritonitisOver time, changes in the peritoneal membrane can lead to alterations in solute transport and loss of ultrafiltration (1). Those changes may necessitate discontinuation of peritoneal dialysis (PD) and transition of patients to hemodialysis.Identifying measurable biomarkers in peritoneal dialysis effluent (PDE) that have the potential to predict the development of membrane damage or failure has been an area of interest for some time. One such marker, cancer antigen 125 (CA125), has been used as a putative measure of the collective mass and health of the mesothelial cells lining the peritoneal cavity.A high molecular weight glycoprotein, CA125 is produced in ovarian cancer cells and is used in monitoring ovarian cancer (2). Human peritoneal mesothelial cells also express CA125, and Zeimet et al. (3) reported that mesothelial cells are actually more potent than ovarian cancer cells in producing it. Visser et al. (4) were the first to suggest that dialysate CA125, measured as an appearance rate at the end of a 4-hour peritoneal equilibration test, could be used as marker of mesothelial cell mass in stable PD patients [reviewed by Krediet (5)]. This theory stood relatively unchallenged for the subsequent decade, and CA125 appearance was used as an outcome biomarker in many studies. It is well accepted that levels of CA125 tend to decline in parallel with the duration of PD (6). Studies of new PD solutions reported effluent CA125 levels as a surrogate outcome of membrane preservation on the assumption that an increase reflects a greater mass of preserved mesothelial cells (7-13).Lai et al. (14) were the first to report the lack of correlation between CA125 levels and the number of mesothelial cells in PDE. Their study differed from others in the way that the PDE was analyzed. They noted a higher percentage of dead cells among the mesothelial cells than among the other cell populations. As a result, they postulated that mesothelial cells in the PDE may partly reflect detachment of damaged cells from the peritoneal membrane rather than surrogacy for mesothelial cell mass as others had speculated.That study was followed with another by Breborowicz et al. (15), who also concluded that CA125 is not a reflection of mesothelial cell mass. Using in vitro cultures of human peritoneal mesothelial cells, those authors measured the CA125 level in PDE and found that it was not correlated with the number of cells in the monolayer of mesothelial cultures. Factors such as the age of the patient and a history of exposure to high glucose content, rather than just the number of mesothelial cells, were found to influence the level of CA125.Interestingly, effluent CA125 can be elevated during episodes of peritonitis (16), certainly not a time typical of optimal mesothelial cell functioning. That observation leads to the question of whether elevated CA125 might reflect mesothelial cell damage or even cell death and slough into the effluent, rather than mesothelial cell health.Taken together, the foregoing observations have resulted in uncertainty about the biologic and diagnostic significance of effluent CA125 (17). Despite considerable research, our current understanding of this biomarker is limited, and the clinical significance of an elevated CA125 level in PDE is unknown.Peritoneal dialysis is challenged by the fact that the very solutions that are used to perform dialysis might actually be causing long-term damage to the peritoneal membrane. It is widely accepted that conventional glucose-based PD solutions contribute to changes in peritoneal membrane structure and function (18). The glucose in conventional solutions is thought to be responsible for deleterious cardiovascular and metabolic effects (19). In addition, the glucose degradation products that are present in heat-sterilized dialysis fluids are known to be responsible for retarding the endogenous process of mesothelial cell repair in vitro (20). That knowledge has led to an emphasis on the development of new solutions that are more “biocompatible” with the peritoneal membrane. A “biocompatible solution” is a heterogeneous term that encompasses various combinations of bicarbonate buffer, neutral pH, and low GDP concentrations, and osmolytes other than glucose.Recent studies of patients treated with various biocompatible solutions have consistently shown that levels of CA125 increase in PDE after exposure—in some cases, in as little as 3 months (7-11). The interpretation is that these solutions may offer an environment that promotes mesothelial cell proliferation and health, with subsequent preservation of the peritoneal membrane. Other studies have also suggested that, compared with traditional solutions, biocompatible solutions may preserve residual renal function (6,11).Any intervention that might prolong the life of the peritoneal membrane and improve patient outcomes would obviously be welcomed by the PD community, and so the excitement surrounding the widespread use of biocompatible solutions around the world is understandable. However, a closer look at the relevant studies also shows that, although effluent CA125 is increased with the use of these solutions, so too is the dialysate-to-plasma (D/P) ratio of creatinine (6) in these patients (6,21-23). That kind of change in transport is usually ascribed to increased effective peritoneal surface area as a result of vasodilatation of the submesothelial blood vessels and is interpreted as a consequence of inflammation. Indeed, other studies have shown that biocompatible solutions are associated with increases in established dialysate markers of inflammation such as interleukin 6 (24).

TABLE 1

Effect of Various Solutions on Level of Cancer Antigen 125 (CA125) and Dialysate-to-Plasma (D/P) Ratio of CreatinineOpen in a separate windowIt has been almost 30 years since the initial studies investigating CA125 as a potential biomarker for mesothelial cell mass. Unfortunately, a solid understanding of the biologic significance of CA125 is still lacking, and many questions remain unanswered. Might it be possible that these solutions cause subclinical inflammation of the peritoneal membrane, leading to the increase in transport status? What if CA125 in PDE actually represents a surrogate marker of inflammation or, as in the case of peritonitis, cell death?The introduction of new biocompatible solutions has also brought new hope for the potential prolongation of the life of the peritoneal membrane, and yet the solutions themselves are surrounded by many unanswered questions. Little is known about their long-term effects. Do they preserve residual renal function? Do they increase mesothelial cell mass? Or are they in fact causing peritoneal inflammation as evidenced by increased transport status? Those important questions need to be answered in future studies before these expensive new solutions are readily adopted. Until then, we must accept the related uncertainty and continue the search for the coveted biomarker of membrane integrity.     

Das Renin–Angiotensin–Aldosteron–System – komplexer als bisher gedacht

ZusammenfassungHintergrund:  Angiotensin II (ANG II) ist ein wichtiger Faktor für das Fortschreiten von Nierenerkrankungen. Neben der bekannten blutdrucksteigernden Wirkung hat ANG II eine Vielzahl von pleiotropen Effekten wie proinflammatorische und profibrogene Wirkungen auf die Niere.Neue Erkenntnisse:   Organe haben lokale ANG–II–generierende Systeme, die vom klassischen endokrinen Renin–Angiotensin–Aldosteron–System (RAAS) völlig unabhängig sind. So können beispielsweise proximale Tubuluszellen der Niere in den Primärharn ANG–II–Konzentrationen abgeben, die bis zu 10 000fach über dem Serumspiegel liegen. Diese lokalen Systeme werden durch Standarddosen von ACE–Hemmern oder AT₁–Blockern nur unvollständig gehemmt. Es gibt neben ACE auch andere Enzymsysteme, die ANG II generieren können. Durch alternative Stoffwechselwege können Peptide erzeugt werden, die wie Angiotensin 1–7 gegenteilige Wirkung im Vergleich zu ANG II haben. Abbauprodukte von ANG II wie Angiotensin IV binden an separate Rezeptoren und können Fibrose initiieren. Die Entdeckung von AT₁–Rezeptor–Dimeren und agonistischen Antikörpern kompliziert das System zusätzlich.Klinische Bedeutung:   Aufgrund der Komplexität des RAAS ist aus pathophysiologischen Überlegungen heraus eine Doppelblockade des Systems mit ACE–Hemmern wie auch AT₁–Rezeptor–Antagonisten sinnvoll. Erste Studien haben gezeigt, dass in bestimmten Risikopopulationen die Progression der chronischen Niereninsuffizienz durch eine solche Doppelblockade im Vergleich zur Monotherapie signifikant verlangsamt werden kann. Dies zeigt, dass neue pathophysiologische Erkenntnisse Eingang in die Klinik finden.