Hypertension. 2004;44:974-981
Published online before print November 15, 2004,
doi: 10.1161/01.HYP.0000149249.09147.b4
(Hypertension. 2004;44:974.)
© 2004 American Heart Association, Inc.
Role of Podocytes for Reversal of Glomerulosclerosis and Proteinuria in the Aging Kidney After Endothelin Inhibition
Jana Ortmann;
Kerstin Amann;
Ralf P. Brandes;
Matthias Kretzler;
Klaus Münter;
Niranjan Parekh;
Tobias Traupe;
Melanie Lange;
Thomas Lattmann;
Matthias Barton
From the Medizinische Poliklinik (J.O., T.T., M.L., T.L., M.B.), Universitätsspital Zürich, Switzerland; Pathologisches Institut (K.A.), Universität Erlangen-Nürnberg, Erlangen, Germany; Zentrum für Kardiovaskuläre Physiologie (R.P.B.), Klinikum der J.-W. Goethe Universität, Frankfurt, Germany; Medizinische Poliklinik (M.K.), Ludwig-Maximilians Universität München, Germany; Cardiovascular Research II (K.M.), Bayer AG, Wuppertal, Germany; and Institut für Physiologie und Pathophysiologie der Universität Heidelberg (N.P.), Germany.
Correspondence to Matthias Barton, MD, Medizinische Poliklinik, Departement für Innere Medizin, Universitätsspital, Rämistrasse 100, CH-8091 Zürich, Switzerland. E-mail barton{at}usz.ch
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Abstract
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The cause of focal-segmental glomerulosclerosis as a consequence
of physiological aging, which is believed to be inexorable,
is unknown. This study investigated whether inhibition of endothelin-1,
a growth-promoting peptide contributing to renal injury in hypertension
and diabetes, affects established glomerulosclerosis and proteinuria
in the aged kidney. We also determined the role of endothelin
receptors for podocyte injury in vivo and in vitro. Aged Wistar
rats, a model of spontaneous age-dependent glomerulosclerosis,
were treated with the orally active endothelin subtype A (ET
A)
receptor antagonist darusentan, and evaluation of renal histology,
renal function studies, and expression analyses were performed.
In vitro experiments using puromycin aminonucleoside to induce
podocyte injury investigated the role of ET
A receptor signaling
for apoptosis, cytoskeletal injury, and DNA synthesis. In aged
Wistar rats, established glomerulosclerosis and proteinuria
were reduced by >50% after 4 weeks of darusentan treatment,
whereas blood pressure, glomerular filtration rate, or tubulo-interstitial
renal injury remained unaffected. Improvement of structural
injury in glomeruli and podocytes was accompanied by a reduction
of the expression of matrix metalloproteinase-9 and p21
Cip1/WAF1.
In vitro experiments blocking ET
A receptors using specific antagonists
or RNA interference prevented apoptosis and structural damage
to podocytes induced by puromycin aminonucleoside. In conclusion,
these results support the hypothesis that endogenous endothelin
contributes to glomerulosclerosis and proteinuria in the aging
kidney. The results further suggest that age-dependent glomerulosclerosis
is not merely a "degenerative" but a reversible process locally
confined to the glomerulus involving recovery of podocytes from
previous injury.
Key Words: arterial presure nephrosclerosis DNA kidney failure renal artery expression kidney renal disease
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Introduction
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Aging represents an important factor determining onset and course
of disease and has become a significant issue in view of the
anticipated increase of the aging population. Aging in humans
and rodents progressively impairs renal function
1,2 and structure,
the latter of which is characterized by damage of podocytes
and mesangial matrix, as well as capillary hypertrophy and obliteration
resulting in glomerulosclerosis.
2 The exact mechanisms underlying
age-dependent renal injury are unknown. In otherwise healthy
individuals

65 years of age, even in the absence of known risk
factors such as hypertension or diabetes, glomerulosclerosis
is frequently present.
3 Currently,

1.4% of the US total population
is affected, and the incidence is expected to increase to >2%
within the next 15 years.
3 Glomerulosclerosis and proteinuria
involve injury of podocytes, also known as glomerular epithelial
cells that maintain an intact filtration barrier and control
glomerular basement membrane turnover under normal conditions.
47 In addition to cell-specific changes during aging, cell cycle
inhibitors such as p21
Cip1/WAF1 have been associated with cellular
senescence and cell growth
8,9 and have been linked to glomerular
injury.
10
Endothelin-1 (ET-1), a mitogen and vasoconstrictor11 signaling via G-proteincoupled endothelin subtype A (ETA) and ETB receptors,12 contributes to growth of glomerular mesangial cells. Expression of ET-1 increases in diseased glomeruli and prevention studies indicate that inhibition of ETA receptors retards the progression of glomerulosclerosis.12 As we have shown previously, ET-1 expression increases in the aging kidney in the absence of other risk factors.13,14 Because podocytes are targets of ET-1,15 we sought to investigate the effects of treatment with an orally active ETA receptor antagonist on renal structure and function in aged rats with established glomerulosclerosis and podocyte injury.
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Methods
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In Vivo Studies
Animal Experiments
Male Wistar rats (IFFA Credo/Charles River) were obtained at
the ages of 1 month and 22 months. At 23 months of age, 8 animals
were euthanized and kidneys removed. At 2 and 23 months, animals
(9 to 10 per group) were randomized to 28 days of treatment
with or without the orally active ET
A receptor antagonist darusentan
(LU135252; 20 mg/kg per day; Knoll AG)
16 administered in the
drinking water.
17 On the day before euthanization, 24-hour metabolic
studies were performed as described.
17 In a subset of animals
(n=6 per group), glomerular filtration and hemodynamics were
determined on the day of euthanization, and the left kidney
was perfusion-fixed at mean arterial pressure by glutaraldehyde
infusion. Studies were in accordance with the institutional
guidelines and approved by the institutional animal care committee.
Blood Pressure, Renal Blood Flow, and Glomerular Filtration Measurements
Measurements of arterial blood pressure and renal blood flow were performed in anesthetized animals using an intra-arterial catheter and a flow probe,18 respectively. Glomerular filtration rate was determined using the single injection technique as described by Hall et al.19 For details, see the online supplement, available at http://www.hypertensionaha.org.
Assessment and Quantification of Renal Injury
Renal damage was as described.20,21 Glomerular volume was determined by measuring glomerular surface area in sections of pressure-fixed kidney by planimetry.20 See online supplement for details.
Glomerular Gene Expression In Vivo
Real-time polymerase chain reaction (PCR) was performed from RNA obtained from laser-dissected glomeruli of the study animals.22 For details, see the online supplement.
Western Blot Analysis and Immunohistochemistry
Western blot analysis of p21Cip1/WAF1 and p27kip1 was performed from frozen renal cortices of the study animals. In situ immunohistochemistry of rat p21Cip1/WAF1 was performed on renal cryosections. See the online supplement for details.
In Vitro Studies
Podocyte Injury
Puromycin aminonucleosideinduced podocyte injury23 was used to investigate the effects of drug treatment and gene silencing.24,25 For cytoskeleton organization experiments, mouse podocytes were used.24 See the online supplement for details.
Gene Expression Analysis
Expression of mRNA in human podocytes was determined as described previously using quantitative real-time PCR.25 See the online supplement for details.
Gene Silencing
RNA interference experiments in human podocytes were performed as described by Tuschl26 after transfection of small interfering RNA (siRNA) or nonsilencing RNA using Lipofectamine 2000 (Invitrogen). See the online supplement for details.
DNA Synthesis
In quiescent cultured human podocytes, DNA synthesis was studied as described previously.27 See the online supplement for details.
Statistical Analyses
Data are means [SD], and n denotes the number of animals or independent in vitro experiments, respectively. Data were analyzed using ANOVA with Bonferroni correction or the MannWhitney U test when appropriate. A P value <0.05 was considered significant.
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Results
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Focal-segmental and global glomerulosclerosis including podocyte
injury, mesangial matrix expansion, capillary occlusion, and
tuft adhesion (
Figure 1a) were present in aged rats and comparable
in animals aged 23 and 24 months (score 0.83 [0.17] and 0.87
[0.11], respectively;
P=0.56). Hypertrophy of the glomerular
capillary basement membrane and podocyte injury (including hypertrophy,
intracellular uptake of proteins/absorption droplets, foot process
fusion, and detachment) were visible by electron microscopy
(
Figure 2a and 2b). Consistent with damage of the podocyte filtration
barrier, aged rats exhibited marked proteinuria (307 [153] versus
51 [10] mg/kg per day;
P=0.0102 versus young;
Figure 3b), with
glomerular filtration rate and serum creatinine levels in the
normal range (
Table). At 23 months of age, rats were assigned
randomly to drug treatment with the ET
A receptor antagonist
darusentan for 28 days. Treatment markedly reduced established
glomerulosclerosis (from 0.876 [0.09] to 0.4 [0.09]; 55% inhibition;
P=0.0008;
Figures 1b and 3
a) and proteinuria (from 307 [153]
to 130 [89] mg/kg per day; 57% inhibition;
P=0.0101;
Figure 3b),
and reversed podocyte injury and glomerular basement membrane
hypertrophy (
Figure 2c and 2d). In aged rats, treatment had
no effect on tubulo-interstitial injury (injury score 1.18 [0.31]
versus 1.26 [0.34];
P=0.7;
Figure 1d), glomerular size (untreated
13 039 [783]; darusentan 12 011 [1895] µm
2;
P=0.178),
plasma renin activity, mean arterial blood pressure, and glomerular
filtration rate (NS;
Table). Darusentan treatment also had no
effect on plasma or urinary creatinine levels (NS;
Table).

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Figure 1. a through d, Representative semithin sections of glomerular changes (top panels) and tubulo-interstitial changes (bottom panels) in untreated and darusentan-treated aged rats. a, Glomerular injury in the absence of treatment. Note moderate mesangial matrix expansion and hypertrophy of podocytes with enlarged nuclei, prominent nucleoles (arrow), and increased number of intracytoplasmatic vesicles (open arrow) compatible with podocyte injury and cell activation. b, Glomerular injury after darusentan treatment. Injury is markedly reduced after treatment, and mesangial matrix expansion and podocyte hypertrophy and activation are much milder. In contrast, tubulo-interstitial changes (ie, tubular atrophy, interstitial fibrosis, and interstitial inflammation) are comparable in untreated (c) and treated (d) animals.
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Figure 2. Representative transmission electron micrographs of podocytes and glomerular basement membranes from untreated (top panels) and darusentan-treated aged kidneys (bottom panels). a, Untreated, showing glomerular basement membrane hypertrophy and podocyte injury and detachment. b, Untreated, high-power micrograph demonstrating thickening of glomerular capillary basement membrane with podocyte detachment. Injury of podocytes is characterized by hypertrophy, inclusion of cytoplasmatic absorption droplets, and diffuse effacement of foot processes. c, Darusentan treatment showing attachment of the podocyte to the basement membrane and reversal of glomerular capillary hypertrophy. d, High-power micrograph showing podocyte recovery after darusentan treatment. Endothelin blockade is associated with a reduction of podocyte injury and regression of absorption droplets. P indicates podocyte.
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Figure 3. Established glomerular injury (a) and proteinuria (b) in aged Wistar rats were reduced after 28 days with the ETA receptor antagonist darusentan. c, Averaged values of Western immunoblots (n=4 per group) p21WAF/Cip1 expression; the age-dependent increase was reversed after darusentan treatment. d, Averaged values of Western immunoblots of p27kip1 protein in young and aged rats (n=4 per group). e, Representative example of p21WAF/Cip1 Western immunoblots from different treatment groups. Protein expression was calculated by OD of blots (c and d). Data are means [SD]. Y indicates young; YLU, young, darusentan; O, old; OLU, old, darusentan. *P<0.05 vs CTL; P<0.05 vs old.
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To determine the effect of treatment on gene and protein expression, we next analyzed expression of the cyclin-dependent kinase inhibitor p21Cip1/WAF1, which also contributes to cell differentiation, senescence, and glomerulosclerosis.9,10 Protein expression of p21Cip1/WAF1 was hardly detectable by Western blot analysis (Figure 3c and 3e) or immunostaining in glomeruli and podocytes of young rats (Figure 4a), whereas expression increased strongly in aged rats (8.5-fold, from 1.8 [1.2] to 14.8 [4.0] optical density [OD] units; P=0.001; Figure 3c and 3e), being particularly localized to glomeruli and podocytes (Figure 4b). After darusentan treatment, expression was markedly reduced (from 14.8 [2.0] to 5.0 [1.2] OD units; 65% inhibition; P=0.006; Figures 3c and 3e and 4
c), and glomerular and podocyte expression of p21Cip1/WAF1 was similar to that in young animals (Figure 4c). Increase of the cyclin-dependent kinase inhibitor p27kip1 expression in aged kidneys was unaffected by darusentan treatment (Figure 3d). We also determined the effects of darusentan treatment for glomerular gene expression in vivo of matrix metalloproteinase-9 (MMP-9), which is involved in glomerular matrix turnover.5 MMP-9 gene expression was determined in laser-dissected glomeruli of the study animals and was found to be reduced by 65% after darusentan treatment (from 6.0 [2.8] to 2.1[1.6] units; n=10 per group; P=0.0015; Figure 5a)

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Figure 4. Immunostaining for p21WAF/Cip1 in renal cryosections stained with hematoxylin/eosin (H&E) showing representative glomerular appearance and glomeruli obtained from young (a), aged untreated (b), and aged darusentan-treated rats (c). Glomerular injury increases podocyte and glomerular p21WAF/Cip1 staining (slight brownish staining visible on purple/blue glomerular H&E staining [b]) that is markedly reduced after darusentan treatment (c) and similar to young controls in which glomerular p21WAF/Cip1 immunostaining is hardly detectable (a).
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Figure 5. a, Gene expression of MMP-9 from laser-dissected glomeruli of aged rats without and after darusentan treatment. Gene expression levels are given as arbitrary units (AU) using the  CT method. b, Effect of ETA receptor blockade on podocyte function in vitro. Puromycin aminonucleoside (50 µg/mL)induced apoptosis in human podocytes (P<0.05 vs control [CTL]). c, Effect of selective ETA receptor antagonists BQ-123 (BQ; 10 nmol/L) or darusentan (LU135252 [LU]; 30 nmol/L) on podocyte MMP-9 gene expression after puromycin injury. d, Effect of ETA receptor RNA interference (siRNA) on podocyte MMP-9 gene expression after puromycin injury; cells in control and puromycin only experiments (20 µg/mL) had been transfected with nonsilencing RNA duplex (ncRNA). e, Effect of ETA receptor siRNA or control experiments using nonsilencing RNA (ncRNA) on de novo DNA synthesis in quiescent human podocytes. f, Effects of ETA antagonists BQ-123 (peptide; 10 nmol/L) or LU135252 (darusentan, nonpeptide; 30 nmol/L) on podocyte cytoskeleton disruption after puromycin injury (10 µg/mL) quantified from pooled mouse podocyte experiments as shown in Figure 6. All PCRs were run in triplicate. O indicates old; OLU, old, darusentan; PAN, puromycin aminonucleoside. *P<0.05 vs CTL; P<0.05 vs PAN alone/old.
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Figure 6. Actin cytoskeleton visualized by actin-phalloidin immunofluorescence (left panels) and corresponding phase-contrast microphotographs (right panels). Representative examples of podocytes in the absence (a and b) or presence of puromycin aminonucleoside (10 µg/mL) alone (c and d) or after pretreatment with ETA receptor antagonists BQ-123 (10 nmol/L; e and f), darusentan (LU, 30 nmol/L; g and h), or the ETB receptor antagonist BQ-788 (10 nmol/L; i and j) are shown. Puromyin-induced cell shrinkage, foot process effacement, and cytoskeleton disruption were largely prevented by the ETA receptor antagonists BQ-123 or LU135252 but not by ETB receptor antagonist BQ-788.
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Because stability and function of podocytes were improved after darusentan treatment in vivo and regulation of calcium signaling by endothelin15 suggests that podocytes are functional targets of ET-1, we set out experiments to investigate the role of endogenous and exogenous endothelin in an in vitro podocyte injury model. Endothelin receptor expression of both endothelin receptor subtypes was present in human podocytes (Ortmann and Barton, unpublished observation, 2004), and MMP-9 gene expression and cytoskeletal disruption were chosen as a read-outs of podocyte injury as described previously.5,23,27 As expected, puromycin injury increased MMP-9 gene expression (507%; n=6 per group; P=0.0041; Figure 5c) and caused podocyte shrinkage and cytoskeleton disruption (Figures 5f and 6c and 6
d).27 MMP-9 gene induction was completely prevented by pretreatment with different ETA receptor antagonists BQ-123 (peptide) and darusentan (nonpeptide), respectively, as well as by RNA interference targeting the ETA receptor (Figure 5c and 5d). The evidence suggesting that ETA receptors contribute to podocyte injury was strengthened further by using recombinant ET-1 (10 nmol/L) as ETA receptor agonist, which increased apoptosis from 100% to 176 [61]% of control (P=0.0021; n=6 per group). Apoptosis was also increased using puromycin aminonucleoside as an unspecific stimulus (from 100% to 314 [86]% of control; n=6 per group; P=0.0021; Figure 5b).
Finally, because animal experiments indicated that blocking endothelin receptors improved podocyte stability and function, we hypothesized that endogenous ET-1 might regulate DNA synthesis in cultured podocytes. ETA receptor RNA interference increased de novo DNA synthesis in cultured human podocytes (from 1523 [81] to 2045 [102] cpm/mg protein; n=11 per group; P<0.0001 versus nonsilencing siRNA duplex; Figure 5e), suggesting that endogenous endothelin suppresses podocyte cell cycle in vitro. This finding is compatible with the increased expression of the cell cycle inhibitor p21Cip1/WAF1 observed in aged podocytes in vivo and its reduction after darusentan treatment.
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Discussion
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Renal aging is associated with renal disease and nonrenal clinical
complications in humans.
3 Using a suitable animal model, we
report partial restoration of glomerular structure in association
with a reversal of proteinuria and glomerular expression of
genes indicative of injury and senescence after ET
A receptor
blockade. Protection from injury was also observed after inhibition
of ET
A receptor signaling in an in vitro model of podocyte injury
using puromycin aminonucleoside. Our results suggest that endothelin
contributes to spontaneous glomerular injury associated with
renal aging. This was found to be a reversible process involving
changes in podocyte structure mediated through ET
A receptor
activation.
The findings reported here were obtained in normotensive animals and independent of blood pressure, glomerular filtration rate, renal blood flow, or tubulo-interstitial changes. Consistent with these results, preliminary data from Rabelinks group indicate that endothelin blockade reduces proteinuria in diabetic patients with a normal glomerular filtration rate. Similar to our study, these investigators found the reversal of established proteinuria to be unrelated to blood pressure, glomerular filtration rate, or renal blood flow.28 The exact mechanisms leading to reversal of established proteinuria after ETA receptor blockade are presently unclear. However, the results presented here strongly support a role for endogenous endothelin and podocyte function, which may be similarly relevant for the development of proteinuria in diabetics.6 Further studies are needed to clarify the exact mechanism(s) by which different drug treatments can reverse glomerular disease.29,30
We have demonstrated previously that aging activates the renal endothelin system.13,14 This activation may be, in part, dependent on angiotensin II.31 Indeed, early studies by Remuzzis group have shown that inhibition of angiotensin II formation with the angiotensin-converting enzyme (ACE) inhibitor perindopril for 4 months slowed development of glomerular and tubulo-interstital injury in aging rats, effects that were accompanied by a pressure-lowering effect of the drug.32 In contrast to these studies, blockade of ET receptors in our experiments had no effect on blood pressure or plasma renin activity, but substantially reversed established glomerulosclerosis and proteinuria without modifying tubulo-interstitial injury within 4 weeks of treatment. These new findings suggest that (1) podocytes can undergo substantial structural recovery after injury has occurred, and (2) podocyte recovery results in selective restoration of kidney structure and function that is locally confined to the glomerulus. Given that previous regression studies observed improvement in tubulo-interstitial injury only after pressure-lowering drug treatment,30 and that in our study, tubulo-interstitial injury and glomerular size were unaffected by treatment, we propose local, pressure-independent mechanisms to be involved in the reversal of glomerulosclerosis, as proposed for the antiproteinuric effects of ACE inhibition.33 Moreover, we observed recently that in mice with focal-segmental glomerulosclerosis, improvement of tubulo-interstitial injury by drug treatment may occur while glomerular injury and proteinuria persist.34 This further suggests independence of disease processes in the glomerulus from those occurring in the tubulo-interstitium.
Although unable to complete cell division, podocytes may re-enter cell cycle and undergo mitosis or nuclear division under certain conditions.5 This ability of podocytes to re-enter the cell cycle would also be supported by the observation that in cultured podocytes, DNA synthesis increased after inhibition of ETA receptors, indicating an inhibitory effect of endogenous endothelin on podocyte cell cycle activity. In the kidney, expression of the cyclin-dependent kinase inhibitor p21Cip1/WAF1 increases with injury and aging.9,10,35,36 Moreover, p21Cip1/WAF1 directly contributes to renal injury because its deletion conveys protection from glomerulosclerosis.10 Indeed, in addition to its role as a cell cycle inhibitor, p21Cip1/WAF1 is involved in cellular senescence and differentiation.8,37 Induction of p21Cip1/WAF1 expression in podocytes occurs in murine and human glomerulosclerosis10,35 and, as shown in the present study, in aged glomeruli. Because we observed a strong reduction of p21Cip1/WAF1 expression after darusentan treatment, structural recovery of renal injury after darusentan treatment may be, at least in part, related to effects of endothelin blockade on cell cycle or cell differentiation during the aging process, which appear to be regulated in a reversible manner.
In the present study, we analyzed gene expression of MMP-9 in glomeruli of the study animals as well as in cultured podocytes, which was found to be downregulated after ETA receptor blockade in vitro and in vivo. MMP-9, also known as gelatinase B, degrades collagen38 and thus contributes to glomerular matrix turnover.5,39 We found normalization of hypertrophy of the glomerular basement membrane in aged rats as detected by electron microscopy after endothelin blockade. Although we did not measure the activity of MMP-9 or other MMPs, or their respective inhibitors,30 these data indicate that ETA receptor blockade has beneficial structural effects on the glomerular capillary basement membrane. The exact mechanisms underlying this observation are yet to be determined. Possibly, stabilization of glomerular and podocyte structure after endothelin receptor blockade may have occurred because of a reduced degradation of mesangial collagen and reduction of glomerulosclerosis recently described for other drugs.29,30 Finally, our observation that podocyte injury was induced by exogenous ET-1 and that injury by the unspecific stimulus puromycin was prevented by ETA receptor blockade indicates that intracellular and extracellular ET-1 contribute to injury. This notion is supported by preliminary data by Morigi et al, who observed induction of ET-1 gene expression and cytoskeleton disruption in mouse podocytes in response to indirect injury after exposure to protein overload in vitro.40
In summary, the results support the hypothesis that endogenous endothelin contributes to glomerulosclerosis and proteinuria in the aging kidney. Our results further suggest that age-dependent glomerulosclerosis is not merely a degenerative but a locally confined reversible process enabling podocytes to recover from previous injury. If operative in humans, reversal of glomerular disease by endothelin inhibition could improve treatment of established renal diseases and their clinical complications in patients.
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Acknowledgments
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This work was supported by the Swiss National Foundation (SCORE
32-58421.99 and 3258426.99/1), the DeutscheForschungs-gemeinschaft
(Am 93/2-3, Br 1839/1-1, Kr 1492/6-1, and SFB 423/Project B
8), the Hanne Liebermann Stiftung, Zürich, and the Kurt
und Senta Hermann Stiftung, Liechtenstein. We thank E. Ammann
and F. Krötz for technical help.
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Footnotes
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At the time of the in vivo studies, Dr Münter was an employee
of Knoll AG, Ludwigshafen, Germany (now Abbott Pharmaceuticals).
Received September 7, 2004;
first decision September 16, 2004;
accepted October 21, 2004.
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