Environmental DNA metabarcoding for benthic monitoring: A review of sediment sampling and DNA extraction methods.

Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring.

Enzymatic and cellular responses in relation to body burden of PAHs in bivalve molluscs: a case study with chronic levels of North Sea and Barents Sea dispersed oil.

Mytilus edulis and Chlamys islandica were exposed to nominal dispersed crude oil concentrations in the range 0.015-0.25 mg/l for one month. Five biomarkers (enzymatic and cellular responses) were analysed together with bioaccumulation of PAHs at the end of exposure. In both species, PAH tissue residues reflected the exposure concentration measured in the water and lipophilicity determined the bioaccumulation levels. Oil caused biomarker responses in both species but more significant alterations in exposed C. islandica were observed. The relationships between exposure levels and enzymatic responses were apparently complex. The integrated biomarker response related against the exposure levels was U-shaped in both species and no correlation with total PAH body burden was found. For the monitoring of chronic offshore discharges, dose- and time-related events should be evaluated in the selection of biomarkers to apply. From this study, cellular damages appear more fitted than enzymatic responses, transient and more complex to interpret.

Bioaccumulation of polycyclic aromatic compounds: 1. Bioconcentration in two marine species and in semipermeable membrane devices during chronic exposure to dispersed crude oil.

Assessing the fate in marine biota of hydrocarbons derived from oil particles that are discharged during exploration and production is of relevant environmental concern. However, a rather complex experimental setup is required to carry out such investigations. In this study, a sophisticated tool, the continuous-flow system (CFS), was used to mimic dispersed oil exposure to marine biota. Polycyclic aromatic hydrocarbon (PAH) uptake was studied in two species, the blue mussel Mytilus edulis and juvenile of the turbot Scophthalmus maximus, and in semipermeable membrane devices (SPMD) exposed to crude oil dispersed in a flow-through system. After an exposure period of 8 to 21 d, elimination in organisms and devices was analyzed for 9 to 10 d following transfer to PAH-free seawater. Principal component analysis (PCA) revealed different PAH patterns. In mussel and SPMD, the PAH profiles were very close to that analyzed in seawater. Slight differences were, however, indicated for large molecules with log Kow above six. Nonachievement of steady-state concentration and bioavailability of PAH in oil droplets may account for these differences. The PAH composition in fish revealed only congeners with two to three aromatic rings. A combination of bioavailability and efficient metabolism of the larger PAH molecules may explain this pattern. The CFS made possible a better understanding of some critical factors governing bioconcentration in marine biota from dispersed oil. Yet the results illustrate that uptake of PAH from exposure to oil particles is complex and that different species may bioconcentrate different molecules depending on factors like life style and metabolic capability to degrade the potential harmful substances. Hence, risk assessment of the actual impact of discharges to marine biota should consider these essential biological and ecological factors.

Bioaccumulation of polycyclic aromatic compounds: 2. Modeling bioaccumulation in marine organisms chronically exposed to dispersed oil.

Within the frame of a large environmental study, we report on a research program that investigated the potential for bioaccumulation and subsequent effect responses in several marine organisms exposed to chronic levels of dispersed crude oil. Body burden can be estimated from kinetic parameters (rate constants for uptake and elimination), and appropriate body burden-effect relationships may improve assessments of environmental risks or the potential for such outcomes following chronic discharges at sea. We conducted a series of experiments in a flow-through system to describe the bioaccumulation kinetics of polycyclic aromatic hydrocarbons (PAH) at low concentrations of dispersed crude oils. Mussels (Mytilus edulis) and juvenile turbot (Scophthalmus maximus) were exposed for periods ranging from 8 to 21 d. Postexposure, the organisms were kept for a period of 9 to 10 d in running seawater to study elimination processes. Rate constants of uptake (k1) and elimination (k2) of the PAHs during and following exposure were calculated using a first-order kinetic model that assumed a decrease of the substances in the environment over time. The estimated bioconcentration factor was calculated from the ratio of k1/k2. The kinetic parameters of two-, three-, and four-ring PAHs in mussel and fish are compared with estimates based on hydrophobicity alone, expressed by the octanol-water partition coefficient, Kow (partitioning theory). A combination of reduced bioavailability of PAHs from oil droplets and degradation processes of PAHs in body tissues seems to explain discrepancies between kinetic rates based on Kow and actual kinetic rates measured in fish. Mussels showed a pattern more in compliance with the partitioning theory.

PAH metabolites in bile, cytochrome P4501A and DNA adducts as environmental risk parameters for chronic oil exposure: a laboratory experiment with Atlantic cod.

In order to perform environmental risk assessments with regard to oil contamination in the sea, it is important to obtain knowledge about threshold levels for possible adverse effects in marine organisms. With this objective in mind, selected biomarkers were studied in Atlantic cod (Gadus morhua) chronically exposed to mechanically dispersed crude oil. The fish were exposed for 30 days in a continuous flow system to nominal concentrations of 0.06, 0.25 and 1 ppm North Sea crude oil. Fish were sampled five times during the exposure period. In addition, the 1 ppm group and the control group were sampled 1 week after the end of exposure. Polyaromatic hydrocarbon (PAH) concentrations in the seawater were analysed regularly by direct fluorescence and, at one occasion, by gas chromatography with mass spectrographic detection (GC/MS) measurements. Liver samples were analysed for parent PAH levels by means of GC/MS measurements, and PAH metabolites in bile were analysed by means of fixed wavelength fluorescence. Cytochrome P450 induction in liver was estimated by ethoxyresorufin-O-deethylase (EROD) activity, and hepatic DNA adducts were analysed by the 32P-postlabelling assay. The parent PAH concentrations in liver showed peak levels 3 days after the start of exposure, followed by a reduction towards the end of the experiment. In contrast, the PAH metabolites in bile and EROD activity showed generally increasing levels throughout the whole exposure period, indicating an increased biotransformation efficiency. The level of DNA adducts in the 1 ppm group showed a stable increase during the entire exposure period. Only a slight, non-significant decrease in DNA adduct levels was observed after 7 days of recovery in clean water. Exposure-dependent responses were observed for all three biomarkers. The lowest nominal concentration of dispersed oil in water, 0.06 ppm, corresponded to a measured total PAH concentration in the water of 0.3 ppb. Atlantic cod exposed to this concentration showed increased levels of PAH metabolites in bile and a slight induction of CYP1A, as well as formation of DNA adducts when compared with control fish. Particularly noteworthy is the detection of DNA adducts at such a low exposure concentration of oil in water, which, to our knowledge, is a novel finding. These dose-response data may serve as useful contributions when assessing environmental risk with regard to marine oil pollution.

Outer membrane protein A (OmpA) binds to and activates human macrophages.

Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (P40), we have analyzed the interaction between OmpA and macrophages. We report that Alexa488-labeled P40 binds (at 4 degrees C) to murine and human macrophages in a dose-dependent manner and is rapidly internalized (at 37 degrees C). No binding or internalization of the Alexa488-labeled glycophorin A control protein is observed under the same conditions. Furthermore, P40 up-regulates the production of IL-1beta, IL-8, IL-10, IL-12, and TNF-alpha by human macrophages and of NO by the RAW 264.7 murine macrophage cell line. P40 also synergizes with IFN-gamma and suboptimal concentrations of LPS to up-regulate the production of these mediators. In conclusion, P40 binds to and activates macrophages. These data suggest that recognition of OmpA by macrophages may be an initiating event in the antibacterial host response.

OmpA targets dendritic cells, induces their maturation and delivers antigen into the MHC class I presentation pathway.

We analyzed the interaction between a bacterial cell wall protein and dendritic cells (DCs). Outer membrane protein A from Klebsiella pneumoniae (kpOmpA) specifically bound to professional antigen presenting cells and was endocytosed by immature DCs via a receptor-dependent mechanism. kpOmpA signaled through Toll-like receptor 2, induced DCs to produce interleukin 12 and induced maturation of DCs. Whole antigen that was coupled to kpOmpA and injected into mice was taken up by DCs and delivered to the conventional cytosolic MHC class I presentation pathway. kpOmpA also primed antigen-specific CD8+ CTLs in the absence of CD4+ T cell help or adjuvant and elicited therapeutic immunity to antigen-expressing tumors. Thus, OmpA belongs to a class of proteins that are able to elicit CTL responses to exogenous antigen.

Carrier properties of a protein derived from outer membrane protein A of Klebsiella pneumoniae.

We have recently cloned a new protein, recombinant P40 (rP40). When tested in vivo after conjugation to a B-cell epitope, rP40 induces an important antibody response without the need for adjuvant. To characterize its potency, this carrier protein was coupled to a peptide derived from respiratory syncytial virus attachment G protein (G1'). After immunization of mice with the rP40-G1' conjugate, strong antipeptide antibodies were detected, whereas peptide alone was not immunogenic. To emphasize the carrier properties of rP40, a polysaccharide derived from Haemophilus influenzae type b (Hib) was coupled to it. Immunoglobulin G responses against the Hib polysaccharide were observed after coupling to rP40. Interestingly, an antipeptide antibody response was observed despite preexisting anti-rP40 antibodies generated by preimmunization with rP40. In addition, rP40 compares well with the reference carrier protein, tetanus toxoid (TT), since antibody responses of equal intensity were observed when a peptide or a polysaccharide was coupled to TT and rP40. Moreover, rP40 had advantages compared to TT; e.g., it induced a mixed Th1/Th2 response, whereas TT induced only a Th2 profile. Together, the results indicate that rP40 is a novel carrier protein with potential for use as an alternative carrier for human vaccination.

The recombinant Klebsiella pneumoniae outer membrane protein OmpA has carrier properties for conjugated antigenic peptides.

Klebsiella pneumoniae OmpA, the 40-kDa major protein of the outer membrane, was cloned and expressed in Escherichia coli. The recombinant protein was produced intracellularly in E. coli as inclusion bodies. Fusion of a short peptide to the N-terminus of native P40 facilitated high-level expression of the recombinant protein. Purified recombinant P40 was analyzed to verify purity and structural integrity. The molecular mass of purified recombinant P40 determined by electrospray mass spectrometry was 37,061 Da, in agreement with the theoretical mass deduced from the DNA sequence. Specific proliferation of recombinant-P40-primed murine lymph node cells in response to recombinant P40 stimulation in vitro indicated the presence of a T-cell epitope on recombinant P40. The induction of high serum antibody titers to a synthetic peptide derived from the attachment protein G of the respiratory syncytial virus when chemically coupled to recombinant P40 indicated that the protein had potent carrier properties.

Chromosomal sequencing using a PCR-based biotin-capture method allowed isolation of the complete gene for the outer membrane protein A of Klebsiella pneumoniae.

By employing a novel biotin- and PCR-assisted capture method, which allows determination of unknown sequences on chromosomal DNA, the gene for the outer membrane protein A (OmpA) of Klebsiella pneumoniae has been isolated and sequenced to completion. The method involves linear amplification of DNA from a biotinylated primer annealing to a region with known sequence. After capture of the amplified single-stranded DNA on to paramagnetic beads, unspecifically annealing primers, i.e. arbitrary primers, were used to generate sequences with only partly determined nt sequences. The homology of the sequenced gene to ompA of related bacteria is discussed, and the gene fragment was assembled for intracellular expression in Escherichia coli, and two different fusion proteins were produced and recovered with good yields. The importance of the novel chromosomal sequencing method for gene isolation in general and the potential use of the OmpA fusion proteins are discussed.

Angiotensin-converting enzyme inhibition does not suppress plasma angiotensin II increase during exercise in humans.

Physical effort stimulates the reninangiotensin system (RAS). We studied the effect of an angiotensin-converting enzyme inhibitor (ACE inhibitor) in a double-blind placebo-controlled study, on eight volunteers undergoing physical stress on an ergometric bicycle. The effects of captopril (C) (50 mg, three times daily for 3 days) on arterial pressure (AP), O2 consumption (VO2), variations in auricular natriuretic factor (ANF), renin, angiotensin II (AII) plasma levels, as well as glomerular filtration rate (GFR) and microalbuminuria (MA) were evaluated. The different parameters were compared by analysis of variance (ANOVA). The pressure profile and VO2 were not modified by ACE inhibitor. Exercise stimulates release of renin; this action was greater with captopril administration (treatment effect: p < 10(-4), indicating blockade of the RAS. This inhibition was incomplete because AII levels increased markedly when captopril was given (no treatment effect: p < 0.37). Finally, ACE inhibitor resulted in decreased GFR (p = 115 +/- 5.8 ml/mn-1, C = 91.1 +/- 4, p < 0.05) with exercise without modification of MA. ACE inhibitor administration does not modify the physical performance of nonathletic subjects; AII is significantly increased with exercise despite captopril treatment; ACE inhibitor decreases GFR significantly but does not influence MA with prolonged physical effort.

Biochemical and physiological studies on two T-kininogen species using monoclonal antibodies.

Rat T-kininogens were characterized in plasma, urine and liver slice incubation medium in different physiological conditions using monoclonal antibodies that allow to distinguish between the two forms of T-kininogen. T-kininogen purified from the plasma of both normal and inflamed Wistar and Sprague-Dawley rats was found to contain the two forms of T-kininogen, TI and TII, separated by non-denaturing polyacrylamide gel electrophoresis. The two forms were also found in the plasma of several strains of normal and inflamed rats, except in the plasma of the Buffalo rat which contained only TII-kininogen. The two forms of T-kininogen were also found in the media in which liver slices from inflamed and non-inflamed wistar rats had been incubated. The urine T-kininogen of normal rats was chiefly TI-kininogen, but both forms were found in the urine of inflamed rats. T-kininogen in the plasma of normal and inflamed rats was further characterized by chromatography on Con A-Ultrogel. In normal plasma, we observed a ratio of non-retained to retained T-kininogen of 0.41. The retained T-kininogen was eluted as two peaks, one eluted with 45 mM and the other with 120 mM alpha-methyl-D-glucoside. The ratio of non-adsorbed to adsorbed T-kininogen in inflamed Wistar rat plasma was 1.40 and the retained material was almost exclusively in a single peak, which eluted with 50 mM alpha-methyl-D-glucoside. The non-adsorbed and adsorbed fractions contained both forms of T-kininogen, but the protein bands in the non-retained fraction had greater mobilities on non-denaturing PAGE. SDS-PAGE analysis of T-kininogen deglycosylated by N-glycosidase F showed a major band with a molecular mass of 50 kDa, whereas the molecular mass of the native form was 66 kDa. We concluded that both forms of T-kininogen exist in the non-inflamed and the inflamed rat plasma, except for the Buffalo rat, and that T-kininogen displays a different chromatographic pattern on Con A-Ultrogel after inflammation suggesting altered glycosylation.

Hypertension in Dahl salt-sensitive rats: biochemical and immunohistochemical studies.

1. The renin-angiotensin and kinin-kallikrein systems of Dahl salt-sensitive and salt-resistant rats fed diets with different salt contents were analysed using biochemical and immunocytochemical techniques. 2. Blood pressure increased by 45% in salt-sensitive rats only, after 4 weeks on a high-salt diet. The plasma renin activity and plasma angiotensin II concentration remained at the same levels in salt-sensitive rats on the high-salt diet as on the normal salt diet, whereas the plasma renin activity and plasma angiotensin II concentration of salt-resistant rats fed the high-salt diet were lower. The plasma renin activity and the plasma angiotensin II concentration were elevated in both salt-resistant and salt-sensitive rats fed the salt-deficient diet but were much more elevated in salt-resistant than in salt-sensitive rats. 3. The kidney immunocytochemical data paralleled the data on plasma parameters. Salt-sensitive rats had fewer renin positive juxtaglomerular apparatuses than salt-resistant rats on the normal diet, and the increase on the sodium-deficient diet was also smaller in salt-sensitive rats. Salt-sensitive rats fed the high-salt diet and the standard diet had almost no angiotensin II immunoreactivity compared with the salt-resistant rats on the same diets. 4. The total renal kallikrein content of salt-sensitive rats was lower than that of salt-resistant rats on all three diets, as was the amount of kallikrein excreted in the urine on the standard and the high-salt diets. The differences resulted from a reduction in active kallikrein. The increase in kallikrein in salt-sensitive and salt-resistant rats on the salt-deficient diet was not significantly different. 5. There were similar changes in immunopositive kallikrein in the kidneys of salt-sensitive and salt-resistant rats with diet, with a large increase in kallikrein biosynthesis on the low-salt diet. The plasma concentration of high-molecular-mass kininogen was not significantly different in salt-sensitive and salt-resistant rats, but there was a significant increase in T-kininogen in salt-sensitive rats fed the high-salt diet. 6. In conclusion, the absence of decreases in the plasma renin activity and the plasma angiotensin II concentration in salt-sensitive rats fed the high-salt diet might partially explain the increase in blood pressure.

Comparative study of asparagine-linked glycans of plasma T-kininogen in normal rats and during acute inflammation.

Rat T-kininogen has been separated into two molecular variants by affinity chromatography on concanavalin A (ConA): a ConA-reactive (ConA+) and a ConA-non-reactive (ConA-) fraction, from which carbohydrate chains were quantitatively released by hydrazinolysis. On the basis of high-resolution 400 MHz 1H-n.m.r. spectroscopy of the re-N-acetylated hydrazinolysates, the carbohydrate structures of the two ConA molecular variants of rat T-kininogen were established. The ConA-non-reactive species contains a single type of carbohydrate chain with the following structure: [formula: see text] The ConA-reactive fraction contains the same structure and the following additional one: [formula: see text] The relative abundance of the two molecular forms is profoundly affected during inflammation (ratio ConA+/ConA-: 44% in normal and 95% in inflamed T-kininogen), but no structural modification of the carbohydrate chains was observed.

Immunological characterization of rat kininogens with monoclonal antibodies to T-kininogen. Distinction between the different domains of T-kininogen and the multiple rat kininogens.

A panel of 16 monoclonal antibodies (mAb) were produced against rat T-kininogen to characterize this family of proteins. These mAbs bound 125I-T-kininogen by radioimmunoassay as well as reacting strongly with immobilized T-kininogen in an enzyme-linked immunosorbent assay (ELISA). The reactivity of these antibodies with proteolytic fragments of T-kininogen demonstrated the recognition of several different epitopes. One antibody was specific for the domain 1 of the heavy chain and/or the light chain, twelve antibodies were specific for domain 2 and three antibodies were specific for domain 3. All monoclonal antibodies recognized the two forms of T-kininogen encoded by the two different T-kininogen genes, TI and TII kininogen, except antibody TK 16-3.1 which uniquely reacted with TII kininogen. Two antibodies recognizing domain 2 cross-reacted with the high-molecular-mass kininogen (H-kininogen), whereas all the other monoclonal antibodies were specific to T-kininogen and did not recognize the heavy chain of H-kininogen. None of the antibodies tested altered the thiol protease inhibitory activity of T-kininogen, its partial proteolysis by rat mast cell chymase or the hydrolysis of H-kininogen by rat urinary kallikrein. The use of these antibodies in the development of sensitive ELISA to measure T-kininogen levels in plasma, urine, liver microsomes and hepatocytes is described. Two different forms of T-kininogen were distinguished by these monoclonal antibodies in Western blotting using rat plasma. The localization of T-kininogen was defined using these monoclonal antibodies by immunohistochemistry in rat liver hepatocytes and rat kidney.

Immunocytochemical localization of albumin, transferrin, angiotensinogen and kininogens during the initial stages of the rat liver differentiation.

Rat albumin, transferrin, angiotensinogen, T kininogen (TKg) and high molecular weight kininogen (HKg) gene expression was examined immunocytochemically in embryonic and fetal livers. All these plasmatic proteins, angiotensinogen excepted, are detected as early as day 11 of gestation in intestine epithelial cells and embryonic hepatocytes. Angiotensinogen becomes expressible only at day 13 of gestation. During the early fetal period, the protein immunostaining increases strikingly in parallel with the hepatocyte differentiation. Albumin and transferrin are highly expressed comparatively to kininogens and angiotensinogen. For the first time, specific HKg is demonstrated in the rat liver.

The kallikrein-kinin system in the rat hypothalamus. Immunohistochemical localization of high molecular weight kininogen and T kininogen in different neuronal systems.

High molecular weight kininogen (HKg) and T kininogen (TKg) were detected and localized by immunocytochemistry in adult rat hypothalamus. In addition, kininogens were measured by their direct radioimmunoassay (RIA) or by indirect estimation of kinins released after trypsin hydrolysis and high pressure liquid chromatography (HPLC) separation of bradykinin (BK) and T kinin. A specific HKg immunoreactivity demonstrated with antibodies directed against the light chain (LC) of HKg was colocated with SRIF in neurons of hypothalamic periventricular area (PVA) projecting to external zone (ZE) of median eminence (ME). Heavy chain (HC) immunoreactivity which could be related to HKg or to low molecular weight kininogen (LKg) was detected in some other systems: i) parvocellular neurons of suprachiasmatic (SCN) and arcuate nuclei containing SRIF, ii) magnocellular neurons (mostly oxytocinergic) of paraventricular (PVN) and supraoptic (SON) nuclei, iii) neurons of dorsomedian and lateral hypothalamic areas. TKg immunostaining was restricted to magnocellular neurons of PVN, SON, accessory nuclei (mostly vasopressinergic) and to parvocellular neurons of SCN (vasopressinergic). TKg projections are directed towards the internal zone (ZI) of ME, but very few immunoreactive terminals are detectable in neurohypophysis. TKg staining parallels with vasopressin during water deprivation, and is undetectable in homozygous Brattleboro rats. In some magnocellular neurons, TKg and HC (related to HKg or LKg) are coexpressed. TKg, was also detected in hypothalamus and cerebellum extracts by direct RIA, and BK and T kinin were identified after trypsin hydrolysis. HKg and LKg can act as precursor of BK which can play a physiological role as releasing factor, neuromodulator--neurotransmitter,--or modulator of local microcirculation in hypothalamus. The three kininogens are also potent thiolprotease inhibitors which could modulate both the maturation processes of peptidic hormones and their inactivation and catabolism.

Effect of bradykinin and kininogens in isolated rat kidney vasoconstricted by angiotensin II.

The hemodynamic and endocrine effects of bradykinin and kininogens were investigated using a closed-circuit isolated rat kidney perfused with angiotensin II (ANG II). ANG II induced vasoconstriction, stimulation of urinary kallikrein release, and inhibition of renin secretion. Bradykinin markedly increased renal perfusate flow (RPF) and produced a slight but significant diuresis and natriuresis. The inhibitory effect of ANG II on renin secretion was delayed. Urinary kallikrein secretion was unchanged. The effect of bradykinin was suppressed by the competitive kinin antagonist [DArg,Hyp3,Thi5,8,DPhe7]bradykinin. Kallikrein-sensitive rat high-molecular-weight kininogen produced a progressive rise in renal perfusate flow. Exocrine function and renin and kallikrein secretions were unchanged. Immunoreactive kinins, identified as bradykinin by high-pressure liquid chromatography, were liberated into the perfusate. Perfusate immunoreactive high-molecular-weight kininogen decreased in parallel as a result of consumption. The kalikrein-resistant T-kininogen was not hydrolyzed to release a kinin, had no effect on renal function, and its concentration in the perfusate remained constant. These results suggest that kinin can be produced in the renal circulation from kallikrein-sensitive circulating kininogens and can antagonize the vasoconstrictor effect of ANG II and alter renal hemodynamics. They provide evidence that the kallikrein-kinin system can participate with the renin-angiotensin system in the control of renal blood flow.

The angiotensin converting enzyme in the kidney.

Immunohistochemical studies and experiments with microdissected nephron segments indicate that the angiotensin I converting enzyme (ACE) in the kidney is expressed in the vascular endothelial cells of the renal vessels and in the epithelial cells of the proximal convoluted tubule and the pars recta. Angiotensin converting enzyme is a membrane-bound zinc metallopeptidase and the primary structure has recently been determined by protein sequencing and molecular cloning. It is probably anchored to the cell membrane by a single, short, transmembrane domain located near the carboxy-terminal extremity. The larger, externally situated, amino-terminal part of the molecule is organized in two large, highly homologous domains, each with a putative active site. The function of the endothelial enzyme in the renal vessels is primarily related to angiotensin II (Ang II) formation. However, its level of expression in renal vessels, especially at the glomerular level, appears to be very low in the adult human kidney, and there is evidence that the conversion of angiotensin I (Ang I) may be a rate-limiting step in Ang II formation in the kidney. The vascular enzyme may also contribute to the inactivation of kinins in the peritubular circulation. In the epithelial cells of the proximal tubule, ACE is present in both the brush border and the basolateral membrane. Although the basolateral enzyme may be involved in Ang II formation in the peritubular interstitium, the function of the enzyme on the brush border is unknown. The effects of ACE inhibitors on renal function are primarily, if not exclusively, related to Ang II suppression and perhaps kinin potentiation in the renal circulation.