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*
Department of Anatomy, Charité, Humboldt
University, Berlin, Germany
Department of Nephrology, University of Freiburg, Freiburg,
Germany
Department of Nephrology, University of
Göttingen,
Göttingen, Germany
Department of Clinical Chemistry,
Rheinisch-Westfälische Technische Hochschule
Aachen, Aachen, Germany
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Department of Pharmacology, Ruhr University, Bochum, Germany.
Correspondence to Dr. Sebastian Bachmann, AG Anatomie/Elektronenmikroskopie, Charité CVK, BMFZ, Augustenburger Platz 1, D-13353 Berlin, Germany. Phone: +49-30-450-528-411; Fax: +49-30-450-528-922; E-mail: sbachm{at}charite.de
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1ß1 sGC
was studied with a novel, highly specific antibody against the ß1
subunit. In parallel, the presence of mRNA encoding both subunits was
investigated by using in situ hybridization and reverse
transcription-PCR assays. The NO-induced, sGC-dependent accumulation of cGMP
in cytosolic extracts of tissues and cells was measured in vitro.
Renal glomerular arterioles, including the renin-producing granular cells,
mesangium, and descending vasa recta, as well as cortical and medullary
interstitial fibroblasts, expressed sGC. Stimulation of isolated mesangial
cells, renal fibroblasts, and hepatic Ito cells with a NO donor resulted in
markedly increased cytosolic cGMP levels. This assessment of sGC expression
and activity in vascular and interstitial cells of kidney and liver may have
implications for understanding the role of local cGMP signaling cascades. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit (73 to 88 kD) and one ß subunit (70 kD)
(for review, see reference 1).
The 1ß1 isoenzyme is thought to be the major form. In addition,
two other subunits, 2 and ß2, have been cloned, and an
2ß1 isoform has been functionally characterized
(2,3).
sGC is the most well characterized receptor for nitric oxide (NO); binding of
L-arginine-derived NO to the heme group of sGC results in marked stimulation
of the enzyme, thus increasing the intra-cellular cGMP concentration
(4). Increases in cGMP levels
are responsible for cellular events that ultimately lead to decreases in
intracellular calcium concentrations and smooth muscle relaxation
(5) or to regulation of
multiple genes through interactions with their respective promoters
(6).
In the kidney, both vascular and tubular effects of NO have been observed
(for review, see references 7,
8, and
9), and the cellular sources
for constitutive NO synthase (NOS), which catalyzes the formation of NO, have
been identified
(10,11).
Because of the wide diversity of cell types in this organ, a detailed
knowledge of sGC distribution is required for an understanding of local,
cGMP-mediated effects of NO. A number of earlier studies demonstrated the
organ- and cell-specific presence of sGC mRNA, by PCR and Northern blot
analyses
(12,13,14,15),
and of the immunoreactive protein, by immunohistochemical and Western blot
analyses
(16,17).
To date, however, there is still disagreement with respect to the reported
immunohistochemical distribution of sGC and functional data. This study was
performed in kidneys, to test the hypothesis that more structures than
previously established are involved in NO-sGC-cGMP signaling as a major
regulatory pathway in end-organ perfusion and specific cell function. We used
a newly generated, affinity-purified antibody against a carboxyterminal domain
of ß1 sGC, which, because of its monospecificity in Western blotting
analyses and its cellular localization spectrum, was clearly superior to
previously used antisera. Immunohistochemical data obtained with this antibody
were corroborated by Western blot analyses of extracts from tissues and
isolated cell preparations, by in situ hybridization, by reverse
transcription (RT)-PCR assays of tissues and isolated cell extracts (using
probes for both 1 and ß1 sGC), and by in vitro assessment
of NO-dependent accumulation of cGMP in tissues and cultured cells. The liver
was studied to compare cell type specificity of sGC localization and function
with another end-organ with a well established role of the NO-sGC-cGMP
pathway. Mechanisms involved in NO-dependent regulation of local hepatic
microcirculation were previously identified
(18,19,20).
Common aspects of cGMP-dependent signaling in kidney and liver are
discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation of Glomeruli
Glomeruli were isolated as described previously
(21). As for all other tissue
isolation procedures described in this section, rats were anesthetized with
sodium pentobarbital (50 mg/kg body wt). Kidneys were removed and prepared,
under sterile conditions, in ice-cold RPMI 1640 medium (Seromed, Berlin,
Germany). Cortices were sieved by using steel sieves with pore sizes of 150
µm and then 100 µm. The sieved structures were captured with a smaller
sieve (pore size, 50 µm), transferred to 50-ml tubes, and centrifuged (4000
rpm, 4°C, 10 min). The pellet was then transferred into a small volume (2
to 3 ml) and divided into aliquots (100 µl). The aliquots were transferred
to a microdissection chamber, 200 glomeruli/aliquot were microdissected, and
aliquots were pooled and centrifuged (15,000 rpm, 1 min, 4°C). The
resulting pellet was then prepared for mRNA extraction.
Isolation of Renal Mesangial Cells and Podocytes
Mesangial cells were isolated and cultured as described previously
(22). In brief, glomeruli were
obtained as described above, incubated with collagenase (1 g/L; Sigma,
Deisenhofen, Germany) for 15 min, and suspended in RPMI 1640 medium
supplemented with 170 g/L fetal calf serum, 2.5 mM L-glutamine, 0.1 mM sodium
pyruvate, 100 U/ml penicillin, 100 mg/L streptomycin, 0.2 g/L nonessential
amino acids (all from Seromed, Berlin, Germany), and 5 mg/L
insulin-transferrin-sodium selenite supplement (Roche, Mannheim, Germany).
Approximately 50 glomeruli/cm2 were plated onto collagencoated
glass coverslips (Greiner, Nürtingen, Germany)
and incubated at 37°C in an incubator with a water-saturated atmosphere of
5% CO2/95% air. Mesangial cells were morphologically characterized
by phase-contrast microscopy. They stained positively for smooth muscle actin,
desmin, and vimentin but not for cytokeratin and factor VIII, which
demonstrates the absence of glomerular epithelial and endothelial cells. Cells
responded to 10-4 mM angiotensin II with increases in free
cytosolic calcium concentrations.
Podocytes were isolated and cultured as described
(23). Briefly, immortalized
mouse podocytes carrying the thermosensitive variant of the SV40 T antigen
inserted into the mouse genome were used. These podocytes proliferate at
33°C in the presence of interferon 
, whereas cells are transformed
into the quiescent differentiated phenotype at 37°C in the absence of
interferon . Podocytes then stain positively for the podocyte
differentiation markers WT-1 and synaptopodin. Cells between passage 14 and
passage 20 were seeded at 37°C onto collagen-coated plates and cultured
for at least 7 d, until cells were differentiated, in standard RPMI 1640
medium containing 10% fetal calf serum, 100 U/ml penicillin, and 100 mg/L
streptomycin.
Isolation and Culture of Renal Medullary Fibroblasts
Detailed procedures for the isolation and culture of rat inner medullary
fibroblasts were published previously
(24). In brief, rats were
euthanized by cervical dislocation. Kidneys were immediately removed, and the
inner medulla was excised. Tissue was placed in 290-mosmol, ice-cold,
Hepes-Ringer's buffer (118 mM NaCl, 16 mM H-Hepes, 16 mM Na-Hepes, 14 mM
glucose, 3.2 mM KCl, 2.5 mM CaCl2, 1.8 mM MgSO4, 1.8 mM
KH2PO4, pH 7.4), minced with a razor blade, and
subsequently incubated for 75 min at 37°C in Hepes-Ringer's buffer
containing 0.2% (wt/vol) collagenase (CLS II; Cooper, Frankfurt, Germany) and
0.2% (wt/vol) hyaluronidase (Roche Diagnostics, Mannheim, Germany). After
completion of the incubation procedure, the majority of the collecting duct
cells in suspension were removed by low-speed centrifugation. The supernatants
from the first two low-speed centrifugations, containing the majority of
interstitial cells, were further separated from collecting duct cells with the
use of beads coated with Dolichos biflorus agglutinin, as described
(24). The resulting cell
suspension was then subjected to single-step density gradient centrifugation
with Nycodenz (Nyegaard Co., Oslo, Norway). After centrifugation, interstitial
cells were maximally enriched, with a density of 1.081 to 1.093
g/cm3. After removal of the Nycodenz, cells were plated in culture
wells and maintained in Dulbecco's modified Eagle's medium/nutrient mixture
Ham's F-12 medium (1:1) supplemented with 2 mM glutamine, 1 mM sodium
pyruvate, 1% (vol/vol) nonessential amino acids, 50 U/ml penicillin, 50 U/ml
streptomycin, and 10% fetal calf serum (all from Life Technologies,
Eggenstein, Germany). Passage 1 cultures were examined.
Isolation and Culture of Hepatic Ito Cells
The isolation and culture of liver Ito cells from male Sprague-Dawley rats
(body weight, 500 to 600 g) were performed as described previously
(25). In brief, nonparenchymal
liver cells were isolated by using the pronase-collagenase method
(26). Ito cells were purified
by single-step density gradient centrifugation in Nycodenz (see above) and
were identified on the basis of their typical light-microscopic appearance and
vitamin A-specific autofluorescence. The mean purity of freshly isolated cells
was 90 ± 5%, cell viability was >95%, and the yield ranged from 30
to 50 x 106 cells/liver. Ito cells were seeded at a density
of 0.2 x 106 cells/cm2, in 2 ml of Dulbecco's
modified Eagle's medium containing 4 mM L-glutamine, 10% fetal calf serum,
1000 U/ml penicillin, and 100 mg/ml streptomycin. Cells were maintained in a
humidified atmosphere of 5% CO2/95% air at 37°C. The medium was
changed approximately 20 h after seeding, after which the purity of Ito cells
was >97%. The second medium change was approximately 28 h after seeding, at
which time fetal calf serum supplementation was reduced to 0.2%.
Histochemical and Western Blotting Protocols
Primary Antibodies. A polyclonal antibody against the
carboxy-terminus of the ß1 subunit (SRKNTGTEETEQDEN) of bovine lung sGC
(27) was raised in rabbits and
immunopurified using the antigenic peptide coupled to SulfoLink coupling gel
(Pierce, Boston, MA). A rabbit polyclonal antibody against NOS1 purified from
porcine cerebellum (28) was a
gift from Bernd Mayer (Graz, Austria). A mouse monoclonal antibody against the
podocyte-specific antigen podosynapsin was kindly provided by Peter Mundel
(New York, NY). A mouse monoclonal antibody against human -smooth
muscle actin was acquired from Dako (Glostrup, Denmark). A mouse monoclonal
antibody against human desmin was also acquired from Dako. A rabbit polyclonal
antibody against ecto-5'-nucleotidase was a gift from Brigitte Kaissling
(Zurich, Switzerland).
Western Blot Analyses. Freshly isolated kidneys, lungs, skeletal muscle, and liver from rats were rapidly dissected and cut into small pieces. The cortex and medulla from kidneys were separated. Isolated Ito cells, mesangial cells, and interstitial cells were also assayed. These samples were homogenized on ice in homogenization buffer [175 mM NaCl, 1 mM ethylenediaminetetraacetate, 50 mM triethanolamine-HCl, pH 7.4, 2 mM dithiothreitol (DTT), 1 µM pepstatin, 0.2 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride], using a glass/glass homogenizer. The homogenate was then centrifuged for 30 min at 4°C at 200,000 x g. The supernatant (cytosol) was supplemented with 50% (vol/vol) glycerol and stored at -20°C. Cytosolic proteins (16 to 20 µg) were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (7.5%) and blotted onto nitrocellulose membranes. Blots were blocked for 30 min with Roti-Block (Roth, Karlsruhe, Germany) and incubated overnight at 4°C with antibody against ß1 sGC. After extensive washing, blots were incubated with a horseradish peroxidase-linked anti-rabbit IgG (Sigma). Immunoreactive bands at 70 kD were detected on the basis of chemiluminescence, using an enhanced chemiluminescence kit (Amersham Pharmacia, Freiburg, Germany). In blots generated from extracts of the renal cortex, a degradation band of approximately 55 kD was detected.
Immunohistochemical Analyses. Immunolabeling was performed with cryostat sections of 3- to 5-µm thickness. After being blocked with 5% skim milk in PBS (pH 7.4), sections were incubated with primary antibody for 2 h at room temperature and then overnight at 4°C. The different primary antibodies were administered simultaneously in double-labeling experiments. Thorough rinsing in PBS was followed by signal detection with Cy3-conjugated goat anti-rabbit IgG serum (diluted 1:250 in skim milk-PBS) and Cy2-conjugated donkey anti-mouse IgG (diluted 1:100), for 1 h at room temperature (all secondary antisera from Dianova, Hamburg, Germany). In double-labeling experiments, secondary antibodies coupled to different fluorochromes were applied. Control experiments to confirm the specificity of the antibody against ß1 sGC were performed with omission of specific antibody, as well as competition with the antigenic peptide.
Ultrastructural Pre-Embedding Histochemical Analyses. For fine structural immunolabeling and immunoperoxidase labeling, an established protocol was used (10); for incubation of 20-µm-thick slices generated with a Vibratome, anti-ß1 sGC antibody was used at dilutions between 1:25 and 1:50. Sections were incubated overnight in microtiter plates, postfixed with 1% osmium tetroxide, rinsed in maleate buffer, stained en bloc with uranyl acetate, and flat-embedded in Epon 812. Semithin sections were produced and photographed by using a light microscope. Ultrathin sections were then cut and viewed by using an electron microscope. Control experiments were performed by replacing primary antibodies with skim milk-PBS controls.
NADPH-Diaphorase Staining. The catalytic activity of NOS was demonstrated by enzymatic reduction of nitro blue tetrazolium in the presence of NADPH (NADPH-diaphorase reaction) (28). Slides were washed in PBS and incubated for 15 to 20 min in 0.1 M phosphate buffer containing 0.3% Triton X-100, 0.01% nitro blue tetrazolium, and 0.1% NADPH. No reaction product was observed when NADPH was replaced by NADH.
In Situ Hybridization. The mRNA expression of the 1
and ß1 subunits of sGC was investigated by in situ hybridization
using digoxigenin-labeled riboprobes made from the bovine cDNA coding for the
respective subunits. According to the protocol provided by the manufacturer
(Roche), sense and antisense riboprobes were generated by in vitro
transcription of the 647-bp 1 or 2000-bp ß1 sGC cDNA fragment,
using T3 and T7 polymerases and digoxigenin-labeled UTP, followed by
time-controlled alkaline hydrolysis. For in situ hybridization,
7-µm cryostat sections were treated according to an established protocol
(10). Briefly, 10 ng sGC
antisense mRNA/µl hybridization mixture was incubated for 18 h at 48°C.
The slides were washed sequentially with decreasing concentrations of SSC at
40°C and then with buffer 1 (0.1 M Tris-HCl, 0.15 M NaCl, pH 7.5) at room
temperature and were then incubated for 30 min with buffer 1 containing 1%
blocking reagent and 0.5% bovine serum albumin. Sheep
anti-digoxigenin-alkaline phosphatase conjugate (diluted 1:500 in blocking
medium) was applied for 60 min at room temperature and then overnight at
4°C. The slides were washed twice with buffer 1 and rinsed in buffer 3
(0.1 M Tris-HCl, 0.1 M NaCl, 0.05 M MgCl2, pH 9.5). A solution of
4-nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolylphosphate, and
levamisole dissolved in buffer 3 was then used for the color reaction. The
reaction was stopped by two washes with buffer 4 (0.1 M Tris-HCl, 1 mM
ethylenediaminetetraacetate, pH 8.0). As a control, sense probes were applied
in parallel with antisense probes. Slides were rinsed with PBS and
coverslipped with PBS-glycerol.
RT-PCR
Total RNA was isolated from kidney cortex, medulla, and liver by using a
commercially available kit (InViTek, Berlin, Germany). RNA was extracted with
phenol/chloroform, precipitated with isopropanol, and resuspended in
diethylpyrocarbonate-treated water. RNA from isolated glomeruli was extracted
by using the guanidinium thiocyanate method
(29). All samples were
quantified by spectrophotometric analyses at 260 nm. Five micrograms of total
RNA from each sample were reverse-transcribed with 60 U of murine Moloney
leukemia virus reverse transcriptase for 25 min at 37°C, in a total volume
of 15 µl, according to the protocol provided by the manufacturer (Roche).
The sample were then heated at 70°C for 5 min, to inactivate the enzyme.
These cDNA were used to compare the amounts of 1 sGC mRNA or ß1
sGC mRNA versus glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA
in different tissues. PCR were performed with specific primers for 1
sGC (5'-CCACATCAACACCGGCTAAT-3' and
5'-GAAGTGCAAGDTTCAGTCTC-3'), for ß1 sGC
(5'-CGGATGCCACGGTATTGTCT-3' and
5'-CTCCTGGCTTGACGCACATT-3'), and for GADPH
(5'-TATCCGTTGTGGATCTGAC-3' and
5'-TGGTCCAGGGGTTTCTTAC-3' or
5'-ACCACAGTCCATGCCATCAC-3' and
5'-TCCACCACCCTGTTGCTGTA-3'). cDNA fragments of the expected sizes
of 331 bp (1 sGC), 329 bp (ß1 sGC), and 304 and 446 bp (GAPDH with
the two different primer pairs used) were amplified in 35 cycles (20 s at
94°C, 20 s at 59°C/63°C, and 30 s at 72°C) with 0.06 U/ml
Taq polymerase and 50 mM MgCl2. The PCR products were
separated on 3% agarose gels, stained with ethidium bromide, and observed with
ultraviolet illumination.
Quantitative RT-PCR
Quantitative RT-PCR is based on the assumptions that the cDNA template and
a competitive internal template compete equally for the primers and that
amplification is colinear. The PCR fragment obtained with the ß1 sGC
primers (see above) contains two sites for the restriction enzyme
NlaIII. Digestion of the ß1 sGC PCR product with NlaIII
yielded three fragments, which were separated on agarose gels and then
purified. The outer two fragments were ligated and served as a competitive
template. A 0.3-µg sample of total RNA was reverse-transcribed in the
presence of decreasing amounts of the internal standard (20 ng, 2 ng, 200 pg,
20 pg, and 2 pg), in 25 µl. Each assay included 0.06 U/ml Taq
polymerase (InViTek), 50 mM MgCl2, 25 mM dNTP, 0.1 mM DTT, and 10
mM concentrations of the aforementioned ß1 sGC primers. The cDNA were
amplified in 30 cycles (20 s at 94°C, 20 s at 59°C, and 30 s at
72°C), size-fractionated in 3% agarose gels, stained with ethidium
bromide, and observed with ultraviolet illumination.
Determination of sGC Activity in Cytosolic Fractions by RIA
Cytosolic proteins (10 µg each) from the indicated tissues were
incubated in the presence of 300 µM GTP, 3 mM MgCl2, 3 mM DTT,
0.5 mg/ml bovine serum albumin, 0.25 g/L creatine phosphokinase, 5 mM creatine
phosphate, 1 mM 3-isobutyl-1-methylxanthine (RBI,
Köln, Germany), and 50 mM triethanolamine
hydrochloride (pH 7.4), in a total volume of 0.1 ml. Stimulation of sGC was
performed by addition of 300 µM S-nitrosoglutathione (Alexis,
Grünberg, Germany). The incubation was stopped
by the addition of ice-cold ethanol (final concentration, 70%). Formed cGMP
was measured by RIA, as described
(30).
Measurements of Intracellular cGMP Levels in Isolated Cells
Cells were cultured in six-well plates, maintained at 37°C, and rinsed
with physiologic Ringer's solution. After preincubation with 0.5 M
3-isobutyl-1-methylxantine for 5 min, cells were exposed to
S-nitroso-N-acetylpenicillamine (SNAP) (100 µM and, in
the case of podocytes, 1000 µM; Biomol, Hamburg, Germany) for 30 or 60 min.
In control experiments, 1H-(1,2,4)oxadiazole[4,3-a]quinoxalin-1-one
(10 µM; Alexis) was added simultaneously with SNAP for 30 min. For
termination of the assay, the supernatants were rapidly removed and cells were
rinsed with ice-cold 70% ethanol. After ethanol extraction, cGMP
concentrations were measured with an enzyme-linked immunosorbent assay
(Amersham Buchler, Braunschweig, Germany). To confirm the specificity of the
cGMP pathway, we tested atrial natriuretic peptide (ANP) (1 µM; Sigma),
which stimulates the membrane-bound guanylyl cyclase, for 30 min.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 and ß1 subunits (Figure
3b). Intraglomerular structures did not reliably exhibit an in
situ hybridization signal, which may be attributable to insufficient
sensitivity of the method used. A weak signal was observed in the
extraglomerular mesangium, sometimes in continuity with reactive portions of
the glomerular arterioles.
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In the cortical interstitium, fibroblasts were labeled throughout the
cortical labyrinth, the medullary rays, and the perivascular areas. The
immunoreactive cells were identified by double-labeling with an antibody
directed against ecto-5'-nucleotidase, an enzyme that is typically
located along the cell membranes of cortical fibroblasts
(Figure 4, a and b).
sGC-immunoreactive fibroblasts were further identified throughout the outer
medulla and along the vascular bundles extending to the inner medulla.
Ultrastructural immunoperoxidase staining revealed intense, evenly
distributed, cytosolic ß1 sGC staining in these cells, sparing all major
organelles (Figure 4c). In
situ hybridization also produced strong 1 and ß1 sGC mRNA
signals in peritubular and perivascular locations, with a distribution pattern
analogous to that typical of interstitial fibroblasts
(Figure 4d).
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In the renal medulla, the descending vasa recta demonstrated continuous
strong ß1 sGC immunoreactivity within the contractile media along their
initial portions; in the terminal portions, the remaining pericytes, which
were typically arranged around the circumference of these vessels, were
positively stained (Figure 5).
Strong labeling in pericytes was particularly evident in the vascular bundles
of the outer and inner stripes of the outer medulla. In the inner medulla, the
number of immunoreactive cells progressively decreased toward the papillary
tip. Immunoreactive perivascular fibroblasts in the vascular bundles were
identified on the basis of their characteristic branched morphologic features,
which are distinct from those of vascular pericytes
(Figure 5a). Ascending vasa
recta were not regularly accompanied by ß1 sGC-immunoreactive cells. The
locations of signals produced by in situ hybridization with probes
for the and ß subunits of sGC corresponded to the
immunoreactivity distribution. We detected mRNA signal primarily in
perivascular pericytes and fibroblasts of the vascular bundles (outer
medulla), whereas interstitial fibroblasts in the inter-bundle regions were
rarely labeled. In the inner medulla, only a few interstitial cells were
positively stained.
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Localization of sGC in Liver
In the liver, hepatic stellate (Ito) cells expressed significant levels of
ß1 sGC immunoreactivity and 1/ß1 sGC mRNA, as revealed by
in situ hybridization (Figure 6, a
and b). In the periphery of a hepatic lobule, nearly all Ito cells
were intensively immunostained for sGC; the intensity decreased toward the
central vein, however, and no signal was detectable in the innermost region
around the central vein. High-resolution immunoperoxidase labeling permitted
identification of these cells on the basis of their typical perisinusoidal
location in the space of Disse and their regular content of large lipid
vacuoles, which were surrounded by an intense signal for the cyclase
(Figure 6c). The walls of
portal venules and larger veins were also observed to be
sGC-immunopositive.
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The specificity of the immunohistochemical labeling was verified by
preabsorption of the ß1 sGC-specific antibody with the peptide used for
immunization. No signal was observed in kidney or liver after incubation with
this mixture. For in situ hybridization analyses, sense and antisense
probes transcribed from 1 and ß1 cDNA, respectively, were
routinely used on sections. No signals were obtained with the sense
probes.
Measurements of NO-Stimulated cGMP Formation in Extracts from Tissues
and Isolated Cells
Determination of NO-stimulated sGC activity revealed significant increases
in cGMP formation in the cytosolic fractions from different tissues. The
kidney cortex demonstrated an 87-fold increase in cGMP formation (56 ±
53 to 4899 ± 686 pmol cGMP/min per mg)
(Figure 7A) after stimulation
with S-nitrosoglutathione, and the renal medulla demonstrated a
65-fold increase (33 ± 18 to 2133 ± 445 pmol cGMP/min per mg).
We observed a 232-fold increase in the liver (8 ± 6 to 1856 ±
866 pmol cGMP/min per mg) and a 59-fold increase in the lung (112 ± 101
to 6659 ± 650 pmol cGMP/min per mg). The stimulation factors thus
ranged between 56- and 232-fold, demonstrating that the observed rates of cGMP
formation were substantially and significantly enhanced by NO-dependent
activation of sGC and that, in absolute terms, the lung exhibited the highest
levels of cGMP generation, as expected.
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Incubation of extracts from freshly prepared mesangial cells with the NO donor SNAP led to a 39-fold increase in cGMP levels (153 ± 46 to 5922 ± 1168 pmol/well) (Figure 7B), incubation of extracts from renal medullary interstitial cells with SNAP led to a 52-fold increase (27 ± 4 to 1396 ± 366 pmol/well), and incubation of extracts from isolated hepatic Ito cells with SNAP led to a 47-fold increase (26 ± 7 to 1218 ± 177 pmol/well). The cGMP accumulation induced by the NO donor was almost completely inhibited when cells were stimulated in the presence of the specific sGC inhibitor 1H-(1,2,4)oxadiazole[4,3-a]quinoxalin-1-one. Incubation of extracts from cultured podocytes with various concentrations of the NO donor SNAP, however, never produced an increase in cGMP levels, compared with control values; in contrast, ANP produced a significant 39-fold increase (21 ± 17 to 819 ± 112 pmol/well) (Figure 7C).
Western Blot Analyses
Qualitative immunoblotting was performed with anti-ß1 sGC antibody,
using tissue extracts from kidney cortex, kidney medulla, liver, lung, and
muscle and extracts of isolated Ito cells, mesangial cells, and interstitial
cells that had been tested in the cGMP assays described above. A principal
band was identified, with an apparent molecular mass of approximately 70 kD
(Figure 8). Lower-molecular
mass bands were attributable to degradation. The weak signal in extracts from
interstitial cells may be attributable to the origin of these cells from the
renal medulla, where the number of sGC-immunoreactive fibroblasts is smaller
than in the cortex. The immunohistochemical signal obtained with antibody
against ß1 sGC was also weaker.
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RT-PCR Analysis
We performed a quantitative analysis of ß1 sGC mRNA in extracts from
renal cortex, to verify the histochemical results regarding sGC mRNA
expression. Approximately equal amounts of sGC cDNA and internal standard were
amplified at a concentration of 0.02 pg of standard DNA
(Figure 9A). As a result, a
value of 66 pg sGC mRNA/µg total renal cortical mRNA was calculated. The
presence of mRNA for both 1 and ß1 subunits was further assessed
in tissue extracts by using RT-PCR, with GAPDH as a reference standard.
Significant bands for both subunits were observed for RNA extracts obtained
from kidney cortex, isolated glomeruli, and liver
(Figure 9, B to E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and one ß subunit for
catalytic action
(3,27).
Because we were limited to detection of only the ß1 subunit with reliable
immunohistochemical specificity, we supplemented our data with results from
in situ hybridization and RT-PCR assays with specific probes for both
of the "universal" subunits (1 and ß1) and with
findings from functional assays on cultivated cells that were selected
according to their histochemical identification in situ.
The renal distribution of sGC revealed vascular and interstitial
components. This study demonstrates, for the first time, the presence of sGC
in all contractile cells of the kidney, with different expression levels. In
glomeruli, the smooth muscle-derived cells of the intra- and extraglomerular
mesangium were sGC-immunoreactive. RT-PCR data for isolated glomeruli support
the presence of mRNA coding for 1 and ß1 sGC. The intraglomerular
mesangial cells are thought to maintain mechanical strength by anchoring the
capillary loops in the glomerular tuft
(32); receptors for vasoactive
hormones related to the contractility of these cells have been identified (for
review, see reference 33).
Local effects of the NO-sGC-cGMP pathway may counteract these effects. In
vivo, NO may be derived from constitutive NOS activity of the adjacent
glomerular capillary endothelium or may diffuse from the nearby macula densa
(8,10).
Significant sGC staining, as detected, in the mesangial angles anchoring the
glomerular basement membrane
(32) suggests a particular
role for cGMP in maintaining adequate contractile tone at these sites.
Stimulation with a NO agonist was highly effective in increasing cGMP
accumulation in mesangial cells in vitro, which confirms earlier data
(34) and supports the
hypothesis that cGMP may play an important role in these cells.
The histochemical presence of ß1 sGC in mesangial cells and not in
podocytes, as observed in this study, is in contrast to previous findings
reporting 1 sGC immunoreactivity exclusively in podocytes
(31). However, because those
earlier attempts to localize sGC did not reveal a plausible overall
distribution pattern for the enzyme and failed to demonstrate colocalization
of its subunits, the significance of those experiments must be reconsidered in
light of the findings presented here. Our in vitro data demonstrated,
in fact, that addition of a NO donor to cultured immortalized mouse podocytes
failed to stimulate sGC, whereas the same cells demonstrated a response after
stimulation with ANP; these results highlight the absence of sGC but confirm
earlier findings on the presence of the particulate form of the enzyme in
these cells in rats. Of course, discrepancies between the two species with
respect to sGC expression cannot be entirely ruled out by the reported assay
results.
Marked expression of ß1 sGC in cells of the extraglomerular mesangium may have functional implications. Because the mechanical integrity of the glomerular hilus is thought to be supported by these cells forming a "closure device" for the entrance into the glomerulus (35), NO-dependent regulation of their contractility, mediated by the nearby constitutive NOS in the macula densa, seems plausible (36). Extraglomerular mesangial cells are thought to transmit stimuli from the macula densa to the glomerular vasculature and the renin-containing granular cells, and the NO-sGC-cGMP pathway may represent one of the principal components in this information transfer, possibly in conjunction with structurally established intercellular communication via gap junctional coupling (9,36,37).
The findings of significant sGC immunoreactivity and transcript levels in the glomerular arteriolar walls, including the granular cells, confirm functional observations regarding the local effects of NO on these vascular cells (7,8,9,38). Adjustment of tubuloglomerular feedback action in response to changes in tubular NaCl load and macula densa constitutive NOS activity (8,38) may thus be related to the local abundance of sGC. The presence of sGC in the cytosol directly surrounding the renin-containing granules of the renin-producing cells supports a stimulatory role for cGMP (7,9). Prominent sGC immunoreactivity in the muscular wall of the efferent arterioles further suggests that local release of NO in this vascular portion may have important vasodilatory effects. The particularly high sGC signal in juxtamedullary efferent arterioles suggests a NO-dependent dilatory mechanism, which may determine renal medullary perfusion. Such an effect would be supported by the continuous strong sGC immunostaining in the myocytes constituting the wall of the descending vasa recta and in the pericytes of the terminal portions of these vessels within the vascular bundles. These data may represent the morphologic equivalent of the otherwise well established effects of NO on renal medullary circulation (10,11). The overall weaker ß1 sGC immunostaining of larger renal cortical arteries and arterioles, compared with small resistance vessels, suggests preferential responsiveness to NO in the latter.
Significant expression of sGC was also observed in interstitial fibroblasts
in the renal cortex and, to a lesser extent, in the medulla; in situ
hybridization results and the in vitro data on NO-dependent cGMP
formation, using freshly isolated medullary interstitial cells, confirmed the
immunohistochemical findings, demonstrating that this cell type has a
particular role in the NO-sGC-cGMP signaling pathway in renal parenchyma.
These data agree with an earlier report on mRNA expression of 1 and
ß1 sGC subunits in long-term cultivated rat medullary interstitial cells
and on their NO-specific responsiveness
(39). In situ, a
proportion of the cortical interstitial fibroblasts were previously identified
as the main source of erythropoietin
(40). These cells also express
high levels of NADPH oxidase
(41), and they are involved in
the synthesis of extracellular adenosine; however, it remains to be
established whether these products functionally interact and what role local
cGMP release could play.
The complete absence of tubular histochemical labeling in this study is in contrast to previous PCR and immunohistochemical data that reported sGC mRNA in a variety of tubular epithelia and ß2 sGC immunoreactivity in collecting ducts (12,13,31). The discrepancy between these results may be attributable to sensitivity or specificity problems; however, anti-ß1 sGC immunoreactivity was reliably detected in a variety of renal and hepatic cell types, so that tubular expression, at least of ß1 sGC, must be at a low level. Also, we never observed coincident histochemical localization of NOS and sGC, which suggests that NO, as a signal molecule for cGMP generation, acts through paracrine diffusion to its target, rather than by intracellular signaling.
Our results on sGC in the Ito cells of the liver agree with functional concepts of these cells. Ito cells have been described as liver-specific pericytes that are located in the space of Disse and possess long cell processes extending between the sinusoid endothelia and the parenchymal cells (42). They are related to renal cortical interstitial cells, inasmuch as the two cell types share a number of properties. In addition to the common expression of sGC, they are the principal sources of erythropoietin production by the body (43) and both express neutrophil NADPH oxidase and ecto-5'nucleotidase (41) and play a central role in organ fibrosis (25). In response to vasoactive substances such as NO, carbon monoxide, and endothelin, the contractile tone of Ito cells may be adjusted to regulate sinusoidal microcirculation and portal BP. Both locally formed NO and carbon monoxide are thought to target a common biologic effector limb via binding to sGC and generation of cGMP (18,19,20). The identification of Ito cells as a source of abundant sGC expression thus corroborates the evidence of this signaling cascade, which is also confirmed by the effective stimulation of cGMP release by Ito cells, as assayed in vitro. Kawada et al. (19) reported much weaker release of cGMP in response to Ito cell stimulation by a NO donor than we detected; this difference was probably attributable to differences in cultivation times. Total cGMP-forming activity after stimulation with NO was much lower in liver than in lung and kidney cortex. This difference is likely to be related to the fact that the parenchymal hepatocytes were sGC-negative and the Ito cells, which represent only a small proportion of the entire tissue mass, were the sGC-reactive cell type, in conjunction with venous wall cells.
In conclusion, this study presents an entirely new range of cell types expressing sGC in kidney and liver. Compared with previous data, we thus provide a more solid, extended pattern of sGC distribution, with the use of improved histochemical and in vitro methods. Significant expression was localized to vascular wall cells in both organs, with particularly high intensity in the glomerular arterioles and descending vasa recta of the kidney and in perisinusoidal Ito cells. These sites are crucial for the local adjustment of vascular tone and thus of regional end-organ perfusion. Common properties of Ito cells and renal cortical fibroblasts have been emphasized by the results presented here, which may facilitate an understanding of the functional mechanisms that are active in these cell types. The localization of sGC in renin-producing granular cells corroborates functional concepts of the role of cGMP in renin release. Significant mesangial expression of sGC and effective stimulation of cultured mesangial cells by NO suggest a prominent role for cGMP in adjustment of the contractile tone of the glomerular tuft, with possible implications for diseases of the glomeruli.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit of neutrophil NADPH oxidase and ecto-5'-nucleotidase in
kidney and liver. Kidney Int51
: 479-482,1997[Medline]
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) or presence (
) of 300 µM
S-nitrosoglutathione. Data are the means ± SD of three
representative experiments. (B) Enzyme activity in the cytosolic fractions
from various isolated cells was determined in the absence (
