(Hypertension. 1999;34:1265.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Elements of a Paracrine Tubular Renin-Angiotensin System Along the Entire Nephron
From the Departments of Human Genetics (A.R., T.M., H.F.D., K.W., J.-M.L.), Obstetrics and Gynecology (L.Z., K.W.), and Pathology (D.A.T.), University of Utah School of Medicine, Salt Lake City; the Howard Hughes Medical Institute (C.W.C., E.H., S.Z., T.C., J.-M.L.) and the US Department of Veterans Affairs (T.I., D.A.T.), Salt Lake City, Utah; and the Department of Biochemistry (T.I.), Vanderbilt University, Nashville, Tenn.
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Abstract |
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Abstract—The renin-angiotensin system is a major regulator of body sodium, predominantly through the actions of intrarenal angiotensin II of unclear origin. We show that polarized epithelium of the proximal tubule synthesizes and secretes angiotensinogen at its apical side and that the protein can be detected in urine as a function of dietary sodium. Furthermore, we demonstrate that renin is expressed and secreted in a restricted nephron segment, the connecting tubule, also in a sodium-dependent fashion. A paracrine renin-angiotensin system operating along the entire nephron may contribute to long-term arterial pressure regulation by integrating distant tubular sodium-reabsorbing functions.
Key Words: angiotensinogen • kidney • renin • renin-angiotensin system • sodium, dietary
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Introduction |
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Arterial pressure control is linked to fluid volume and electrolyte balance. Regulation of plasma volume as a function of dietary sodium1 is primarily controlled by the renin-angiotensin system (RAS) and its main effector, angiotensin II (Ang II); Ang II is released from angiotensinogen by 2 cleavage steps involving renin and angiotensin-converting enzyme (ACE).2
The short-term effects of Ang II are better understood than its long-term effects. Acute depletion of body fluid volume triggers a vasoconstrictor response mediated by the circulating RAS, involving renin secreted by the juxtaglomerular apparatus (JGA) in the kidney, angiotensinogen from liver, and ACE at the luminal surface of capillary endothelium.
Sustained low-dose infusion of Ang II leads to a progressive long-term rise of arterial pressure due to cumulative sodium retention primarily mediated by direct intrarenal Ang II effects.1 Ang II has been detected at high concentrations in proximal tubular fluid.3 In contrast to plasma renin (36 to 40 kDa), angiotensinogen (61 to 65 kDa) is not filtered through the glomerular membrane. Abundant angiotensinogen mRNA in proximal tubule (PT) epithelium4 strongly suggests local generation of Ang II by an unspecified mechanism. Renin mRNA at this site can be detected only by application of the very sensitive technique of reverse transcriptase—polymerase chain reaction (RT-PCR).5 Exogenous Ang II stimulates the apical sodium-hydrogen exchanger in the PT6 ; it may also stimulate the epithelial sodium channel and other transporters in distal segments of the nephron.7 8
Fundamental questions remain unanswered, however. If intrarenal Ang II directly affects sodium reabsorption, where is it generated and by what mechanism? How is this mechanism regulated in response to sodium? At what sites does Ang II impact on sodium transport along the nephron? What is the mechanism for coordinated regulation of sodium uptake in proximal and distal segments of the nephron?
The experimental observations reported herein may help delineate specific hypotheses to address these questions. We show that polarized monolayers of PT cells secrete angiotensinogen at their apical side and that angiotensinogen transits through the entire nephron, in view of the fact that it can be measured in final urine. Furthermore, we find that renin is expressed in a restricted segment of the nephron, the connecting tubule (CNT). At both sites, the expression of these components varies with dietary sodium. These hormonal components may contribute to the regulation of tubular functions along the entire nephron.
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Methods |
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Generation of Antibodies
Polyclonal antiserum was raised in rabbits against highly purified mouse submaxillary gland renin according to Misono et al9 and Geoghegan et al.10 It recognized submaxillary gland renin (ren-2) in crude extracts and in purified fractions, ren-1 expressed in COS-1 cells, and prorenin from crude kidney lysates. It did not cross-react with purified cathepsin D, total COS-1 lysates, or crude liver extracts. Polyclonal antiserum for mouse angiotensinogen was raised in rabbits against highly purified mouse angiotensinogen (purified as a glutathione-S-transferase fusion protein). Angiotensinogen was purified by glutathione affinity chromatography, glutathione-S-transferase tag removal, anion exchange, and gel permeation chromatography. Antisera were used at 1:400 to 1:1200 dilutions in immunohistochemistry.
Studies of Angiotensinogen Secretion in Cultured
Cell Monolayers
ts-MPT cells were grown on semiporous membranes as
described.11 Angiotensinogen in cell
medium was measured as the release of angiotensin I (Ang I)
in a renin cleavage reaction (2.5 nmol/L renin in 25 mmol/L NaOAc
[pH 6.5], 0.5 mmol/L AEBSF, 0.5 mmol/L 8-hydroxyquinoline,
and 5 mmol/L EDTA). Ang I was measured with a competitive
radioimmunoassay (NEN DuPont). Integrity of the monolayer was verified
by measurement of transepithelial resistance and diffusion of tritiated
mannitol after addition to the apical chamber.
Animal Experiments and Measurements
C57BL/6 mice were placed in metabolic cages
(Nalgene) and subjected to protocols approved by the Institutional
Animal Care and Use Committee. Twenty-four hours before dietary sodium
manipulations, all animals were fasted with free access to water
supplemented with 2% glucose and 0.1% KCl. Amiloride (1 mg/kg),
furosemide (2 mg/kg), or control carrier was injected subcutaneously.
Low-sodium (0.3%) and high-sodium (6%) diets were purchased from
Purina Mills. Blood was collected by cardiac puncture. Spot urine was
collected by bladder puncture. Hemidissected kidneys were
formalin-fixed or snap-frozen in liquid nitrogen. Urine specimens were
collected at 12-hour intervals in tubes containing AEBSF and
8-hydroxyquinoline. Weight and urine volumes were recorded daily.
RNA isolation and RT-PCR were performed according to standard protocols
(QIAGEN). RT-PCR experiments were performed by use of the Access RT-PCR
system (Promega).
In Situ RT-PCR
In situ RT-PCR was performed as described by Ertsey and
Scavo.12 Digoxigenin-labeled PCR product was detected
in situ with an alkaline phosphatase–conjugated anti-digoxigenin
antibody (Roche) and visualized by adding the substrates nitro blue
tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP, Sigma
Chemical Co). Sections were counterstained with PAS.
Immunohistochemistry
Immunostaining was performed according to
standard protocols (DAKO Co). The biotinylated secondary antibody was
detected by use of streptavidin conjugated with horseradish peroxidase
or alkaline phosphatase and visualized with either
3-amino-9-ethyl-carbazole (AEC, Sigma) or NBT/BCIP, respectively.
Sections visualized with AEC were counterstained with hematoxylin
and eosin.
Quantitative Histology
Renin expression in tubular cells was quantified by evaluating
the frequency of renin immunostaining in cross sections
of the distal nephron with the use of the peroxidase reporter enzyme
and AEC chromogen. Two independent investigators blinded to the
experimental conditions scored kidney sections for renin by using a 0
to 4 scale (0 indicated no tubular renin staining; 1, at least 1
positive cell per tubule section; 2, 25% to 50% positive cells per
tubule section; 3, 50% to 75% positive cells per tubule section; and
4, >75% positive cells per tubule section). Concordance was >90%.
Four separate experiments were performed. Group means were compared by
unpaired t tests; a value of P<0.05 was
considered significant.
Cell Immunoblotting
Arcades of CNTs were microdissected and manually isolated after
limited collagenase digestion. Single cells were obtained
by further collagenase treatment (0.5% at 37°C for 10
minutes). Cells were washed, resuspended in 30 µL serum-free
low-sodium DMEM, transferred onto polyvinylidene fluoride
membranes, incubated overnight, and fixed with 4%
paraformaldehyde. Chinese hamster ovary (CHO) cells
expressing mouse renin and human angiotensinogen served as
positive and negative controls. Immunoblotting was
performed as described13 14 by use of anti-mouse renin
antibody, biotinylated anti-mouse IgG (DAKO Corp), and
streptavidin-alkaline phosphatase (DAKO) at 1:500 dilutions. Alkaline
phosphatase was detected by using the chromogen NBT/BCIP (Sigma).
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Results |
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PT Epithelium Secretes Angiotensinogen at Its Apical Side
Renal expression of angiotensinogen was examined in sodium-restricted animals by immunohistochemistry. Staining was observed only in the PT, and the granular appearance of the protein in the vicinity of PAS-counterstained brush borders suggested a secretory process (Figure 1A). To test this hypothesis, confluent monolayers of conditionally immortalized cells of murine PT11 were grown on semipermeable membranes separating apical and basolateral compartments. In monolayers with verified integrity, angiotensinogen was reproducibly detected in the apical but not in the basolateral compartment (Figure 1B). Under our prevailing conditions, angiotensinogen mRNA was detected by Northern blot,11 whereas renin mRNA was too close to the detection limits of RT-PCR to be conclusive (data not shown).
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If angiotensinogen is secreted in tubular lumen, is it present in final urine? Indeed, the protein was detected in the urine of mice and men by Western blot analysis using a specific polyclonal antiserum (data not shown). Native angiotensinogen was measured in 12-hour urine samples of male mice kept in metabolic cages with unrestricted access to food and water, conditions that did not significantly affect body weight and, therefore, total body water. Urinary angiotensinogen was inversely related to dietary sodium (Figure 1C). Native angiotensinogen was also observed in urine specimens of healthy human volunteers (Figure 1D).
Renin Is Synthesized by Principal Cells of CNT
Transit of angiotensinogen through the nephron
reflects elimination and/or delivery to a downstream site of renin
expression. The distribution of kidney renin was examined in C57BL/6
animals, a strain carrying only the ren-1 gene,15 by using
a submaxillary gland renin (ren-2) antiserum. In sodium-restricted
animals, staining was observed unambiguously in cortical clusters of
open tubular segments devoid of PAS counterstain (Figure 2A, 2B, and 2C). As
expected, intense staining was also observed in JGA (Figure 2B, arrow). The specificity of renin staining was confirmed by several
observations. Staining was absent in sections treated with preimmune
rabbit serum (data not shown) or after preincubation with antigen
(Figure 2D). Furthermore, these observations were confirmed by
use of a previously established polyclonal renin
antiserum.16 For each antiserum, renin immunoreactivity
was jointly observed in JGA and cortical tubular segments over the
entire dilution series tested. Immunoreactive renin was also detected
in similar segments of human kidneys by use of anti-human renin
antiserum (Figure 2E).16
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Past the macula densa, the nephron can be subdivided into distinct entities on the basis of anatomic and functional features. Cortical distal segments include distal convoluted tubule, CNT, and cortical collecting duct. We conclude that tubular renin immunostaining is restricted to CNT segments on the basis of the following arguments: (1) staining is not observed in the larger cortical collecting ducts, (2) staining is observed in clusters of tubular sections located in the cortical labyrinth in the immediate vicinity of radial veins (Figure 2A and 2B), and (3) these clusters are only observed in the midcortical labyrinth demarcated by medullary rays. This topographical arrangement is characteristic of the arcades formed by merging CNTs of midcortical and deep nephrons.17
The epithelium of CNT is composed of 2 main cell types, intercalated
cells and principal cells of CNT (CNT cells). Intercalated cells are
subdivided into 2 subtypes, 
and ß; both express
H+-ATPase, whereas only the ß subtype stains
for peanut lectin. Staining of serial sections for renin,
H+-ATPase (Figures 2F and 2G), or
peanut lectin revealed that cells staining for renin did not stain for
H+-ATPase or peanut lectin, suggesting that they
are not intercalated cells. The morphology of renin-positive cells is
consistent with that reported for CNT cells, with a polygonal
appearance, a convex apical side devoid of brush border, and a
centrally located nucleus within an abundant clear
cytoplasm.17 Renin staining predominates at the apical
side of the cytoplasm and in the vicinity of the nucleus.
The hypothesis of local renin synthesis, as opposed to uptake of
filtered renin of systemic origin, was tested by a combination of
microdissection and RT-PCR. Proximal convoluted tubules (Figure 3A and 3B) and glomeruli (Figure 3A and 3C) were readily identified; junctions between
CNTs and either other CNTs or collecting ducts were used to sample CNT
segments of the nephron (Figure 3A and 3D). Renin
amplification products of expected size and sequence were clearly
observed in RNA preparations from the glomerulus independent of dietary
sodium (Figure 3E). An unambiguous signal was also observed in
isolated CNT arcades, particularly in sodium-restricted animals. Only a
minimal signal was noted in PT under sodium restriction. These
observations were reproduced in 4 independent series of microdissection
experiments. Controls included amplification of GAPDH for RNA quality
and -smooth muscle actin to exclude contamination with JGA
components. The specificity of all amplification products was
confirmed by DNA sequencing.
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To confirm renin synthesis in CNT by an independent method, in situ RT-PCR was applied to kidney sections from mice subjected to 16-hour sodium restriction (Figure 4A to 4F). Renin mRNA was unambiguously identified in cortical segments of distal nephron and in cells of afferent arterioles (Figure 4D to 4D and 4E, arrowhead), nor was it detected in the inner or outer medulla (data not shown). Control sections not pretreated with DNase showed uniform staining of all cells in all nephron segments (Figure 4A and 4B); control sections, DNase-treated but not reverse-transcribed, showed no staining (Figure 4C). Specificity was further confirmed by the absence of signal when primers were applied to samples that were not reverse-transcribed. The specificity of the primers used for amplification was validated by DNA sequencing in control RT-PCR experiments. To further ensure that amplification was specific and not the result of primer extension of fragmented genomic DNA, control amplifications were performed from reaction supernatants.
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CNT Cells Secrete Renin
Renin secretion by CNT cells was demonstrated by cell
immunoblotting.13 14 Cells isolated from
microdissected arcades of CNT secreted renin (Figure 5A). CHO cells expressing mouse renin
(Figure 5B) and human angiotensinogen (Figure 5C) served as positive and negative controls, respectively.
Renin secretion was revealed by pericellular halos of immunoreactive
renin.
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Renin Expression in CNT Varies With Dietary Sodium
In subsequent studies, we have used renin
immunostaining to investigate the relation between
dietary sodium and CNT renin after overnight manipulation of tubular
sodium delivery by varying total sodium intake and/or sodium
reabsorption at specific sites through diuretics. Furosemide
inhibits the
Na+/K+2Cl-
transporter upstream from the distal tubule, whereas amiloride, a
specific inhibitor of the epithelial sodium channel,
affects sodium reabsorption in distal segments of the nephron. Because
of signal saturation of renin immunostaining in JGA,
renin expression at this site was estimated by semiquantitative RT-PCR
of total kidney RNA. Renin expression in CNT cells was assessed by
quantitative histology (frequency of CNT cells staining for renin).
Under high sodium, animals exhibited minimal renin staining in CNT and
moderate JGA renin expression (Figure 6A and group 1A in Figure 6D and 6E). By contrast, the combination of high sodium and amiloride
administration led to a marked increase in CNT immunoreactive renin
(Figure 6B and group 1B in Figure 6D and 6E); JGA
renin was significantly decreased (P<0.05). Overnight
sodium restriction led to a marked increase in CNT immunoreactive renin
(Figure 6C and group 2A in Figure 6D and 6E), but
there was no significant change in JGA renin. However, longer periods
of sodium restriction stimulated renin expression in JGA (data not
shown). The combination of sodium restriction and furosemide resulted
in decreased renin expression in CNT, without additional effects on JGA
renin (groups 2B in Figure 6D and 6E). Sodium intake was
monitored by measuring total sodium excretion. Under these experimental
conditions, the treatments were without effect on body weight,
excluding significant variation in total body water.
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Discussion |
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Our observations support the following conclusions: (1) angiotensinogen is synthesized by PT and secreted into tubular fluid, (2) uncleaved angiotensinogen transits through the entire nephron and can be found in final urine, (3) renin is synthesized and secreted by CNT cells, and (4) both proximal angiotensinogen and distal renin expression vary as a function of dietary sodium. Together with filtered renin, these elements of a tubular RAS may be involved in the coordinated regulation of sodium reabsorption at various sites within the nephron.
Angiotensinogen mRNA in PT and its variation with sodium intake has been observed previously in whole-kidney sections.4 Besides confirming these findings, our data characterize the time course of angiotensinogen expression in parallel with that of renin in CNT and with the urinary excretion of angiotensinogen and Ang I. Furthermore, they demonstrate apical secretion of angiotensinogen by polarized epithelium. Previous experiments with primary culture of heterogeneous cell populations from kidney cortex suggested secretion of the protein but could not resolve the directionality of this process.18 Our observations of urinary angiotensinogen confirm prior reports using laboratory animals.19 20 When measured in human subjects, urinary angiotensinogen was used as a clinical indicator of damage to the glomerular membrane,21 because angiotensinogen is normally not filtered.22
Secretion of angiotensinogen at the apical side of cultured monolayers does not by itself demonstrate luminal secretion in vivo. The apical distribution of angiotensinogen secretory granules in PT (Figure 1A) suggests such a process. So does the presence of angiotensinogen in final urine, in a direct correlation with angiotensinogen expression in PT and in an inverse relation with dietary sodium. Taken together, these observations strongly suggest that angiotensinogen is indeed secreted in tubular fluid in this initial nephron segment. If so, filtered renin could act on luminal angiotensinogen to generate Ang II. The significance of Ang II as a major regulator of sodium transport in this segment, in part through its stimulation of the sodium-hydrogen exchanger, NHE-3, is well documented.3 6
Some reports have suggested the existence of an autocrine RAS in PT, in view of the fact that renin mRNA was detected by RT-PCR of total RNA from selected cell populations or from microdissected PT.5 23 24 The expression levels of renin and angiotensinogen at this site are markedly different, however. Angiotensinogen protein appears abundant in epithelium of PT, whereas renin is below the detection level of immunohistochemistry. Likewise, angiotensinogen mRNA is detected by Northern blot of total RNA. By contrast, evidence of a renin transcript at this site escapes even in situ hybridization after RT-PCR, a faint signal appearing only in liquid-phase RT-PCR. The functional significance of the latter observation is unclear, because faint RT-PCR signals can reflect either legitimate or illegitimate transcription. Whereas the reaction between filtered renin and secreted angiotensinogen may be the predominant mechanism of formation of Ang II in tubular fluid of the PT, an autocrine or intracrine local RAS, expressed at a much lower level, may still serve a distinct purpose in the homeostasis of this segment.
Renin immunoreactivity has been occasionally noted in tubular segments of mouse kidney.25 26 27 28 In one case, where the focus of the investigation was on JGA renin, it was dismissed as an experimental artifact.27 In other instances, it was interpreted as nonspecific uptake of filtered renin on the basis of indirect arguments. In the present study, the hypothesis of local synthesis was examined directly. Concordant results obtained in repeated series of 4 independent experiments for each of 2 different methods strongly support local synthesis. The apical distribution of renin immunostaining in tissue sections (Figure 2C) and the demonstration of renin secretion by isolated CNT cells in vitro suggest luminal secretion. The observation of renin in final urine does not in itself settle this issue, however, because filtered renin may in part escape degradation in PT. Taken together, these data suggest that renin secreted by CNT could act on luminal angiotensinogen of PT origin to release Ang I in luminal fluid. The documented presence of both ACE and Ang II receptors in the collecting duct29 30 would allow formation and action of Ang II in distal segments of the nephron. Although little is known about the effect of Ang II in CNT and the collecting duct, one report does suggest that luminal Ang II stimulates amiloride-sensitive sodium transport in the initial collecting tubule of cortical nephrons.7
The distribution of renin immunostaining in CNT arcades is strikingly similar to the pattern of expression of tissue kallikrein in kidney. Although colocalization of renin and kallikrein remains to be established, it has indeed been shown that tissue kallikrein secreted into the distal tubule31 originates in CNT,32 with predominant immunostaining at the apical side of CNT cells in a pattern quite similar to that observed in the present study for renin.33 It is also known that kininogen is synthesized and secreted in tubular lumen by principal cells of the collecting duct, and bradykinin B2 receptors have been reported at the luminal side of this nephron segment.34 The presence of components of both the RAS and the kallikrein-kinin systems in the luminal compartment of CNT and collecting duct suggests a coordinated function. These systems are interrelated not only through the often-described opposite actions of their effectors, Ang II and bradykinin, but also through multiple areas of potential overlap, such as aldosterone response, sodium and potassium balance, renin activation, and peptide conversion through the action of ACE.
Guyton35 has stressed the significance of the pressure-natriuresis relation in the regulation of baseline arterial pressure and the dominant role of intrarenal Ang II in the regulation of sodium balance in response to variation in dietary sodium. The genetics of rare mendelian hypertension, such as Liddle syndrome36 or the syndrome of mineralocorticoid excess,37 confirms experimental physiology by stressing the significance of sodium reabsorption by distal segments of the nephron in arterial pressure regulation. Angiotensinogen of PT origin and renin expressed by CNT may provide a mechanism to coordinate the functions of proximal and distal segments of the nephron in regulation of sodium balance and blood volume homeostasis. It may be through this system that molecular variation in angiotensinogen38 39 affects individual liability to develop essential hypertension, as suggested by a recent transgenic model.40
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Acknowledgments |
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This study was supported by grant HL-45325 from the National Institutes of Health. Dr Rohrwasser was supported in part by a Boehringer Ingelheim Fellowship. Dr Lalouel is an Investigator of the Howard Hughes Medical Institute. Dr Terreros is a US VA investigator. We thank Drs Cathrine Craven, Toshiaki Nakajima, Raoul Nelson, Gordon Williams, and Christoph Westenfelder for their comments on the manuscript. We thank Todd Peterson for computer reconstruction of photographed nephron segments. This article is dedicated to the memory of Roger R. Williams.
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Footnotes |
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Reprint requests to Jean-Marc Lalouel, MD, DSc, Howard Hughes Medical Institute, University of Utah Health Sciences Center, Eccles Institute of Human Genetics, 6th Floor, Salt Lake City, UT 84112.
1 Both authors contributed equally to the study. 
Received September 2, 1999; first decision September 21, 1999; accepted October 4, 1999.
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