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(Hypertension. 2004;44:974.)
© 2004 American Heart Association, Inc.

Scientific Contributions

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

	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

	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.^4–7 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-protein–coupled endothelin subtype A (ET_A) and ET_B receptors,¹² contributes to growth of glomerular mesangial cells. Expression of ET-1 increases in diseased glomeruli and prevention studies indicate that inhibition of ET_A 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 ET_A receptor antagonist on renal structure and function in aged rats with established glomerulosclerosis and podocyte injury.

	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 p21^Cip1/WAF1 and p27^kip1 was performed from frozen renal cortices of the study animals. In situ immunohistochemistry of rat p21^Cip1/WAF1 was performed on renal cryosections. See the online supplement for details.

In Vitro Studies
Podocyte Injury
Puromycin aminonucleoside–induced 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 Mann–Whitney U test when appropriate. A P value <0.05 was considered significant.

	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 3a) 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] µm²; 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).

View larger version (133K): [in this window] [in a new window]   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.

View larger version (126K): [in this window] [in a new window]   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.

View larger version (38K): [in this window] [in a new window]   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.

View this table: [in this window] [in a new window]   Effects of Darusentan Treatment for 28 Days in Young and Aged Rats on Physiology and Renal Parameters

To determine the effect of treatment on gene and protein expression, we next analyzed expression of the cyclin-dependent kinase inhibitor p21^Cip1/WAF1, which also contributes to cell differentiation, senescence, and glomerulosclerosis.^9,10 Protein expression of p21^Cip1/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 p21^Cip1/WAF1 was similar to that in young animals (Figure 4c). Increase of the cyclin-dependent kinase inhibitor p27^kip1 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)

View larger version (54K): [in this window] [in a new window]   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).

View larger version (31K): [in this window] [in a new window]   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.

View larger version (77K): [in this window] [in a new window]   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.

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 6d).²⁷ MMP-9 gene induction was completely prevented by pretreatment with different ET_A receptor antagonists BQ-123 (peptide) and darusentan (nonpeptide), respectively, as well as by RNA interference targeting the ET_A receptor (Figure 5c and 5d). The evidence suggesting that ET_A receptors contribute to podocyte injury was strengthened further by using recombinant ET-1 (10 nmol/L) as ET_A 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. ET_A 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 p21^Cip1/WAF1 observed in aged podocytes in vivo and its reduction after darusentan treatment.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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 Rabelink’s 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 ET_A 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 Remuzzi’s 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 ET_A receptors, indicating an inhibitory effect of endogenous endothelin on podocyte cell cycle activity. In the kidney, expression of the cyclin-dependent kinase inhibitor p21^Cip1/WAF1 increases with injury and aging.^9,10,35,36 Moreover, p21^Cip1/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, p21^Cip1/WAF1 is involved in cellular senescence and differentiation.^8,37 Induction of p21^Cip1/WAF1 expression in podocytes occurs in murine and human glomerulosclerosis^10,35 and, as shown in the present study, in aged glomeruli. Because we observed a strong reduction of p21^Cip1/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 ET_A 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 ET_A 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 ET_A 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.

	Acknowledgments

 
This work was supported by the Swiss National Foundation (SCORE 32-58421.99 and 32–58426.99/1), 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.

	Footnotes

 
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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