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Department of Nephrology, University of Heidelberg, Germany.
Correspondence to Dr. Jürgen Wagner, Department of Nephrology, University Hospital, University of Heidelberg, Bergheimerstrasse 56a, D-69115 Heidelberg, Germany. Phone: +49-6221-911210; Fax: +49-6221-911279; E-mail: Juergen_wagner{at}med.uni-heidelberg.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 g/24 h, n = 10; OD
ratio (ODR), i.e., wild-type/mutant mRNA ratio, 1.81 ± 0.3],
compared with patients with lower-grade proteinuria (<2 g/24 h, n
= 8; ODR, 0.63 ± 0.1; P < 0.01) or control subjects
(n = 9; ODR, 0.57 ± 0.1; P < 0.01). ET-1 gene
expression was significantly higher among patients with higher-grade
proteinuria, compared with patients with lower-grade proteinuria (P
< 0.01) or control subjects (P < 0.05). ET-RA mRNA
expression was not different among the groups. Patients with higher-grade
proteinuria who were receiving angiotensin-converting enzyme inhibitors
exhibited significantly (P < 0.05) lower ET-1 and ET-RB
mRNA expression, which was comparable to that of control subjects. By using
immunohistochemical analyses, an association between proteinuria and
expression of ET-1 and ET-RB in proximal tubular epithelial cells
and of ET-1 in glomeruli was confirmed in the separate cohort of patients with
IgA nephropathy. It is concluded that the increased ET-RB and ET-1
mRNA and protein expression observed in animal models of renal disease is also
demonstrable among patients with renal disease and high-grade proteinuria. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The diverse actions of ET-1 are mediated via at least two receptor subtypes, i.e., ET-RA and ET-RB. The former subtype mediates cell proliferation and vasoconstriction, and the latter presumably mediates vasodilation via nitric oxide stimulation, natriuresis, and ET clearance (11,12,13,14).
In normal human kidneys, ET-1 and ET-R are confined mainly to vascular tissue and to a lesser extent to glomerular structures (15,16,17,18,19). ET-RB are also observed in tubular epithelial cells (20). In human subjects, increased ET-1 expression was observed in several renal diseases, e.g., allograft nephropathy (21,22) and lupus nephritis (23). These results correspond to findings in several animal models of renal disease in which the ET system is activated. Extrapolation from animal models to human disease is somewhat problematic, however, because of some species-specific differences in the ET system. Under basal conditions, more prominent expression of ET-1 is observed in rat kidneys than in human kidneys. A preponderance of ET-RA is noted in rat kidneys, compared with human kidneys, although ET-RB expression is also markedly elevated in rat kidneys under pathologic conditions (24,25). These considerations emphasize the importance of examining human disease.
Of particular interest is the possibility of coactivation of ET-1 and ET-RB in proteinuric models of renal damage (24,25), suggesting activation of the ET system when tubular epithelial cells are exposed to protein overload. To date, however, expression of the components of the renal ET system has not been examined in detail among patients with proteinuric renal disease. These considerations prompted us to examine the expression of ET-1, ET-RA, and ET-RB in renal biopsies from patients with varying levels of proteinuria.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Control biopsy samples were obtained from nine patients under-going tumor nephrectomy (seven men and two women; median age, 60 yr; age range, 54 to 66 yr; systolic BP, 133 ± 2.2 mmHg; diastolic BP, 84 ± 2.8 mmHg). Renal biopsies were obtained at the time of surgery, from sites remote from renal cell carcinoma-bearing tissue. Patients had not undergone thromboembolization of the renal artery before surgery.
Study 2: Immunohistochemical Analyses in Patients with IgA Nephropathy. Sixteen patients (10 men and six women; median age, 35 yr; age range, 19 to 71 yr) with biopsy-proven IgA nephritis were included. The mean protein excretion was 2.3 ± 0.5 g/24 h, and the creatinine clearance was 95.0 ± 13.0 ml/min. Systolic BP was 133 ± 3.5 mmHg, and diastolic BP was 81 ± 2.9 mmHg. Only two patients were receiving antihypertensive treatment (a calcium channel blocker for one patient and a calcium channel blocker plus a diuretic for the other patient); none of the patients was receiving an ACE inhibitor.
Control biopsy samples were obtained from five patients undergoing tumor nephrectomy (three men and two women; median age, 57 yr; age range, 50 to 63 yr; systolic BP, 129 ± 2.0 mmHg; diastolic BP, 82 ± 3.2 mmHg).
Biopsy Technique
Patients underwent biopsies for diagnostic purposes. Written informed
consent was obtained before the biopsies. The study was approved by the local
ethics committee. Biopsies were performed as described elsewhere
(26). In brief, samples were
obtained from the left lower pole under ultrasonographic guidance (Toshiba
Sonolayer; Toshiba Medical Systems, Neuss, Germany), using a Biopty system
(Radiblast AB, Uppsala, Sweden) and an 18-gauge (1.2-mm) needle. Control
samples were obtained immediately after ligation of the renal artery (<10
min of warm ischemia time). The Biopty needle was directed perpendicularly to
the surface, so that sampling conditions were similar to those for standard
percutaneous renal biopsies. Samples were immediately placed in sterile
reaction tubes (Eppendorf, Hamburg, Germany), shock-frozen in liquid nitrogen,
and stored at -80°C until further analysis.
Measurement of Serum Creatinine Concentrations, Creatinine Clearance,
and Urinary Protein Excretion
Serum and urinary creatinine levels were measured with the
Jaffé method, using an Hitachi autoanalyzer
(Hitachi, Frankfurt, Germany). Twenty-four-hour protein excretion was measured
by using a commercial kit (Roche, Basel, Switzerland) based on the biuret
method.
RNA Isolation and Reverse Transcription
The Trizol (Life Technologies, Gaithersburg, MD) method was used for RNA
isolation, according to the recommendations of the manufacturer. Selected
biopsy specimens were checked for degradation of total RNA on 1% agarose gels.
RNA concentrations were determined by spectrophotometric measurements at
wavelengths of 260/280 nm. Reverse transcription was performed as described
elsewhere (27). For each
biopsy, reverse transcription was performed three times and the resulting cDNA
was pooled.
Quantitative PCR Assays
Quantification of specific mRNA was performed essentially as described by
Paul et al. (28) and
Wagner et al. (27).
For each gene, a DNA deletion mutant was cloned
(29). These mutants had the
same sequences as the wild-type genes (with identical primer binding sites)
but with deletions of a maximum of 20%, resulting in shorter amplification
products. Reverse-transcribed RNA (0.1 µg) was used for amplification in
the presence of defined concentrations of DNA deletion mutants, as an internal
standard. The concentration of standard DNA was selected to allow comparable
degrees of amplification of wild-type and mutant genes. Primers used were as
follows: prepro-ET-1, 5'-TGGCTTTCCAAGGAGCTCC-3' (374 to 392 bp,
sense) and 5'-GCTTGGCAGAAATTCCAGC-3' (692 to 710 bp, antisense);
ET-RA, 5'-AGCTCAGCTTCCTGGTTACC-3' (615 to 634 bp,
sense) and 5'-AATTCCCTGAACACGACTCC-3' (1053 to 1072 bp,
antisense); ET-RB, 5'-TTGGAGCTGAGATGTGTAAGC-3' (763 to
783 bp, sense) and 5'-CAGTGAAGCCATGTTGATACC-3' (1367 to 1387 bp,
antisense) (30).
The PCR mixture contained 0.25 mM dNTP (Promega, Madison, WI), 2.5 mM MgCl2, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 80 nM levels of sense and antisense primers (Life Technologies), and 1 U of Taq polymerase (Life Technologies). The thermal profile used consisted of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, performed 30 times for ET-1 and ET-RB and 32 times for ET-RA. In all experiments, possible contamination with genomic DNA was excluded by PCR amplification in the absence of reverse transcriptase. Amplification products were separated by agarose gel electrophoresis and then digitized by using a gel documentation system (Intas, Göttingen, Germany) and Scion Image software (National Institutes of Health, Bethesda, MD). The ratio of the OD of the endogenous cDNA to the OD of the mutant DNA [OD ratio (ODR)] was determined. Each sample was measured in triplicate, in individual PCR assays, for each gene.
ET-1 and ET-RB Immunohistochemical Studies
Staining Protocol. Primary antibodies 1H11 and BH2B10 (a gift from
Schering Company, Berlin, Germany) were raised in mice against human ET-1 and
ET-RB, respectively. The anti-ET-1 antibody demonstrated 100%
cross-reactivity with ET-1, ET-2, and sarafotoxin-6b and 50% cross-reactivity
with ET-3. The anti-ET-RB antibody did not cross-react with
ET-RA.
Cryostat sections (3-µm thickness) were air-dried for 2 h and fixed in acetone for 10 min at 4°C. Nonspecific binding of avidin or biotin to the tissue was prevented with the use of avidin and biotin blocking solutions (Vector Laboratories, Burlingame, CA). Sections were overlaid with 50 µl of primary mouse antibody solution (diluted 1:10) for 24 h at 4°C. Phosphate-buffered saline was used for solutions and washings (10 min). Subsequent incubations were performed at 20°C for 30 min, in a moist chamber. The primary antibody was washed off, and sections were incubated with 50 µl of secondary (link) antibody (biotinylated goat anti-mouse Ig, 1:40). After washing, sections were incubated with streptavidin-peroxidase complex (1:100). Binding of the primary antibody was observed with freshly prepared 3-amino-9-ethylcarbazole and hydrogen peroxide solution. Sections were counterstained with Mayer's hemalum and mounted under glass coverslips. Negative control experiments were performed by incubating control sections with phosphate-buffered saline or normal mouse serum.
Semiquantitative Grading. Immunostaining was graded essentially as described elsewhere (31), as follows: 0, no staining; 1, mild staining; 2, moderate staining; 3, intense staining. The localization of the reaction, i.e., glomeruli or proximal tubule epithelial cells, was recorded. The intensity and extent of staining were taken into account and graded by two separate investigators, in a blinded manner.
Statistical Analyses
Data are expressed as mean ± SEM or median and range as indicated.
Data were analyzed by using the nonparametric Mann-Whitney test, linear
regression, or multivariate ANOVA, as indicated. The null hypothesis was
rejected at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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80 ml/min. The wild-type/mutant mRNA ODR for patients with
creatinine clearance values of 80 ml/min (n = 8) was 1.06
± 0.26, whereas that for patients with creatinine clearance values of
>80 ml/min (n = 10) was 0.47 ± 0.06 (P < 0.05)
(Figure 1a). No significant
correlation between creatinine clearance and ET-1 ODR was noted by ANOVA,
however. Patients (n = 10) with higher-grade proteinuria (2 g
protein/24 h, 4.5 ± 2.4 grs/24 h, mean ± SD) exhibited
significantly higher levels of ET-1 mRNA expression (ODR, 1.00 ± 0.21),
compared with patients (n = 8) with lower-grade proteinuria (<2 g
protein/24 h, 0.8 ± 0.6 grs/24 h, mean ± SD) (ODR, 0.40 ±
0.04; P < 0.01) and control subjects (ODR, 0.55 ± 0.07;
P < 0.05) (Figure
1b). There was a modest correlation between 24-h protein excretion
rate and ET-1 expression (ODR) (r2 = 0.24, P <
0.04). When patients receiving ACE inhibitors were analyzed, ET-1 gene
expression was significantly lower among patients with higher-grade
proteinuria who were receiving ACE inhibitors (n = 5), compared with
patients who were not (P < 0.05)
(Figure 1c).
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ET-RA mRNA Expression. mRNA levels tended to be higher
among patients with higher-grade proteinuria, but the difference did not reach
statistical significance (Figure
2b). No significant differences in ET-RA gene
expression were noted between patients with creatinine clearances of 80 or
>80 ml/min and control subjects (Figure
2a) or between patients receiving ACE inhibitors and those not
receiving ACE inhibitors (Figure
2c).
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ET-RB mRNA Expression. ET-RB mRNA expression
tended to be higher both for patients with creatinine clearances of >80
ml/min and for those with creatinine clearances of 80 ml/min, compared
with non-nephritic control subjects, but this difference did not reach
statistical significance (Figure
3a). Receptor expression was significantly higher for patients
with higher-grade proteinuria, compared with patients with lower-grade
proteinuria (ODR, 1.81 ± 0.30 versus 0.63 ± 0.13;
P < 0.01) or control subjects (ODR, 0.57 ± 0.09; P
< 0.01) (Figure 3b). There
was some correlation between urinary protein excretion and ET-RB
mRNA expression (ODR) (r2 = 0.28, P < 0.03).
The correlation between proteinuria and ET-RB mRNA expression did
not reach statistical significance by ANOVA, by a narrow margin (P =
0.08). Among patients with higher-grade proteinuria, gene expression by
subjects who were receiving ACE inhibitors was significantly lower than that
by subjects without ACE inhibitor treatment (P < 0.05)
(Figure 3c).
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Study 2: Immunohistochemical Analyses for Patients with IgA
Nephropathy
Immunohistochemical Staining for Immunoreactive ET. A negative
control assay for ET-1 demonstrated no significant nonspecific staining in a
biopsy from a patient with IgA nephropathy and high-grade proteinuria
(Figure 4A). Control sections
demonstrated no or only very weak immunoreactivity in glomeruli (endothelial
and capsular epithelial cells), in the vasculature (endothelial and vascular
smooth muscle cells), and in proximal tubular epithelial cells
(Figure 4C). Among patients
with IgA nephropathy, staining was markedly increased in proximal tubular
epithelial cells and was slightly increased in glomeruli (endothelial and
capsular epithelial cells), as well as in vessels (endothelial and smooth
muscle cells) (Figure 4, D and
E). In addition, among patients with high-grade proteinuria,
staining for ET-1 was prominent in mesangial cells
(Figure 4E).
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Among patients with lower-grade proteinuria, expression of ET-1, as indicated by the staining score, was lower in glomeruli than in proximal tubules in the majority of cases. Expression of ET-1 in glomeruli and proximal tubular epithelial cells was significantly greater among patients with higher-grade proteinuria than among patients with lower-grade proteinuria or control subjects (Figure 5A). The correlation between individual proteinuria values and staining scores for proximal tubular epithelial cells was statistically significant (r2 = 0.32. P < 0.02).
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Immunhistochemical Staining for Immunoreactive ET-RB. A negative control assay for ET-RB demonstrated no significant nonspecific staining in a biopsy from a patient with IgA nephropathy and high-grade proteinuria (Figure 4B). In healthy control biopsies, we observed variable, but usually weak, staining for immunoreactive ET-RB in glomeruli (parietal epithelial cells and podocytes) (Figure 4F), in vessels (endothelial and smooth muscle cells), and in some proximal tubular epithelial cells. For patients with IgA nephropathy (even those with lower-grade proteinuria), staining in proximal tubular epithelial cells was increased, compared with control subjects. Furthermore, staining in glomeruli (capsular epithelial cells, podocytes, endothelial cells, and mesangial cells) and in vessels (endothelial and smooth muscle cells) demonstrated a similar pattern, compared with control samples, or was slightly increased (Figure 4, G and H).
In proximal tubular epithelial cells, the staining scores for ET-RB were significantly higher for patients with IgA nephropathy and higher-grade proteinuria than for patients with lower-grade proteinuria or control subjects (Figure 5B). Staining was more widespread, as well as more intense. In glomeruli, immunoreactivity for ET-RB was not different between control subjects and patients with IgA nephropathy (with higher- or lower-grade proteinuria).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In renal biopsies, the concentrations of ET-1 and ET-RB mRNA determined by quantitative PCR were significantly increased, at least for patients with higher-grade proteinuria. In parallel, ET-RA expression tended to be higher, but this did not reach statistical significance. In a separate cohort of patients with IgA nephropathy and different degrees of proteinuria, increased expression of ET-1 and ET-RB protein was observed and the intensity of staining was again related to proteinuria.
The increase in ET-1 and ET-RB mRNA levels was independent of the type of renal disease; specifically, no difference was observed when inflammatory (IgA nephropathy, lupus nephritis, and mesangioproliferative GN) and noninflammatory (diabetic nephropathy, amyloidosis, and membranous GN) types of renal disease were compared. There was no correlation between age and ET-1 expression, as reported previously for plasma and vascular endothelium of healthy subjects (32,33).
For healthy control subjects, immunohistochemical analyses confirmed faint staining for ET in the renal vasculature, in glomeruli, and in proximal tubular epithelial cells (15). Semi-quantitative evaluation demonstrated marked ET peptide expression in both glomeruli and tubular epithelial cells of patients with higher-grade proteinuria. The observation of increased expression in renal tissue is of interest in view of the report by Ohta et al. (4) that indicated that ET excretion was increased among patients with renal disease. Those authors concluded that urinary ET originated from ET expressed in tubular cells (4). The in vitro studies by Zoja et al. (34) provide further support for this hypothesis; exposure of proximal tubular epithelial cells to albumin and other serum proteins increased tubular ET-1 expression and secretion.
An additional argument for increased activity of the ET system in human renal disease involves the concomitant increases in ET-1 and ET-RB expression. Expression of ET-RA was not significantly increased in whole biopsy samples, but the possibility of increased expression of ET-RA in critical compartments certainly cannot be excluded.
Overall, the concomitant increases in ET-1 and ET-RB expression strongly argue for a potential role of the ET system in renal disease. The increase in ET-1 expression, when examined in isolation, may underestimate the degree of activation of the ET system, because ET-RB is upregulated in parallel with, and presumably in response to, ET-1. Upregulation of ET-RB is surprising, because negative feedback inhibition would be anticipated a priori. For example, in failing hearts, Zolk et al. (35) observed increased ET-1 expression but decreased ET-RB expression. Among our healthy control subjects, ET-RB was faintly expressed in glomeruli, vessels, and proximal tubular epithelial cells, confirming the findings reported by Karet et al. (19). In contrast, for patients with higher-grade proteinuria, staining for immunoreactive ET-RB was prominent in proximal tubular cells but was also observed in glomeruli (Figure 4, B and C). This observation is in good agreement with findings in experimental models of renal disease, e. g., aminonucleoside-induced nephrosis (24) or Thy-1 nephritis (25). It is of interest that, in those models, increased ET-RB expression was noted in the proteinuric phase in parallel with increased ET-1 expression, as observed in the biopsies from our patients with renal disease. It has been argued that exposure of tubular epithelial cells to proteins is the major signal for upregulation of ET-1 (36). Recent findings in lupus nephritis, however, suggest predominant glomerular expression (23); therefore, the mechanisms of activation in different glomerular diseases may be more diverse and complex.
The preferential upregulation of ET-RB (compared with ET-RA) may be of interest in another respect. Iwasaki et al. (37) demonstrated ET-RB-mediated autoinduction of ET-1 in rat mesangial cells, and Ong et al. (20) demonstrated similar autoinduction in human tubular epithelial cells. Both groups demonstrated that autoinduction could be blocked by ET-RB antagonists. The role of ET-RB is still unclear. ET-RB-mediated signals have been implicated in vasodilation, nitric oxide and bradykinin release, and sodium transport (11,38,39). Furthermore, Oksche et al. (40) recently demonstrated that ET-1 is cleared by internalization through ET-RB. The net final result of these diverse local ET-1 actions in tubular epithelial cells is currently unknown. It is most unlikely that the upregulation of ET-1 and ET-RB represents the result of generalized activation of protein-loaded tubular epithelial cells, because analysis of additional renal biopsies from patients with similar degrees of proteinuria (data not shown) confirmed our previous observation (27) that, in contrast to ET-RB, angiotensin II (AngII) receptor type 1 mRNA is downregulated. In summary, the aforementioned results are clearly in accordance with recent proposals that the "nephrotoxic" effects of protein in the urine are mediated, at least to a large extent, via activation of the renal ET system (36).
The primary aim of this study was to assess the effects of proteinuria on the renal ET system. In addition, we observed that activation of the renal ET system was less for a limited number of proteinuric patients who were receiving ACE inhibitors. This finding requires a brief discussion; AngII and the ET system interact. In vitro and in vivo studies demonstrated that AngII induces ET-1 expression (41,42,43). Increased plasma and renal ET-1 peptide concentrations were observed in rats treated with AngII infusions (44). AngII also increased the release of ET-1 in human endothelial cell cultures (45). Decreased ET-1 excretion and decreased expression of ET-1 peptide and prepro-ET-1 mRNA were noted after pharmacologic blockade of the renin-angiotensin system (RAS) by ACE inhibitors and/or AngII receptor type 1 blockers, in several experimental models of renal disease (31,46,47,48,49). The effects of blockade of the RAS may depend on the baseline activity of the RAS, because we observed, at best, modest effects in a low-renin model of chronic renal failure (9). In this study, patients with higher-grade proteinuria who were receiving ACE inhibitors exhibited significantly lower renal ET-1 and ET-RB expression, compared with patients with higher-grade proteinuria who were not receiving ACE inhibitors. A local RAS is present in tubular epithelial cells. Tubular epithelial cells are exposed to very high local AngII concentrations (50). When proteinuria is provoked by daily injections of albumin, the local RAS in proximal tubular epithelial cells is activated (51). This observation is consistent with the idea that an "inflammatory phenotype" of proximal tubular epithelial cells is associated with higher local availability of AngII. Our observation of lower renal expression of ET-1 and ET-RB among patients receiving ACE inhibitors does not allow determination of whether these effects result from local or systemic inhibition of the RAS and whether they are mediated by direct actions on the RAS or indirectly, via reduction of proteinuria. In summary, we conclude that increased expression of ET-RB and ET-1, at the mRNA and protein levels, is observed in renal tubular epithelial cells of patients with severe proteinuria.
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Acknowledgments |
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) and patients with IgA GN
and higher-grade proteinuria (
). Staining
scores were as follows: 0, no staining; 1, weak staining; 2, intermediate
staining; 3, strong staining, *P < 0.05 versus
IgA GN with lower-grade proteinuria.