Atrasentan Reduces Albuminuria by Restoring the Glomerular Endothelial 1 Glycocalyx Barrier in Diabetic Nephropathy 2 Running title: Atrasentan reduces albuminuria 3 4 Margien G.S. Boels 1 , M. Cristina Avramut 2 , Angela Koudijs 1 , Martijn J.C. Dane 1 , Dae Hyun 5 Lee 1 , Johan van der Vlag 3 , Abraham J. Koster 2 , Anton Jan van Zonneveld 1 , Ernst van 6 Faassen 1 , Hermann-Josef Gröne 4 , Bernard M. van den Berg 1 , Ton J. Rabelink 1 7 8 1 Einthoven laboratory for Experimental Vascular Medicine, department of Nephrology, 9 LUMC, Leiden University Medical Center, The Netherlands 10 2 Department of Molecular Cell Biology, LUMC, Leiden University Medical Center, The 11 Netherlands 12 3 Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud 13 University Medical Center, Nijmegen, the Netherlands 14 4 Department of Cellular and Molecular Pathology, German Cancer Research Center, 15 Heidelberg, Germany 16 17 Corresponding author: 18 Margien G.S. Boels 19 Leiden University Medical Center , Einthoven laboratory for Experimental Vascular 20 Medicine, department of Nephrology 21 Albinusdreef 2, 2333 ZA, Leiden, The Netherlands 22 Fax: +3171 526 6868, Phone: +3171 526 2148, e-mail: [email protected]23 24 Word count: 4797 25 Number of figures: 6 26 Supplementary Figures: 2 27 28 29 Page 1 of 32 Diabetes Diabetes Publish Ahead of Print, published online March 25, 2016
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Atrasentan Reduces Albuminuria by Restoring the Glomerular Endothelial 1
Glycocalyx Barrier in Diabetic Nephropathy 2
Running title: Atrasentan reduces albuminuria 3
4
Margien G.S. Boels1, M. Cristina Avramut
2, Angela Koudijs
1, Martijn J.C. Dane
1, Dae Hyun 5
Lee1, Johan van der Vlag
3, Abraham J. Koster
2, Anton Jan van Zonneveld
1, Ernst van 6
Faassen1, Hermann-Josef Gröne
4, Bernard M. van den Berg
1, Ton J. Rabelink
1 7
8 1
Einthoven laboratory for Experimental Vascular Medicine, department of Nephrology, 9
LUMC, Leiden University Medical Center, The Netherlands 10 2 Department of Molecular Cell Biology, LUMC, Leiden University Medical Center, The 11
Netherlands 12 3 Department of Nephrology, Radboud Institute for Molecular Life Sciences, Radboud 13
University Medical Center, Nijmegen, the Netherlands 14 4Department of Cellular and Molecular Pathology, German Cancer Research Center, 15
Heidelberg, Germany 16
17
Corresponding author: 18
Margien G.S. Boels 19
Leiden University Medical Center , Einthoven laboratory for Experimental Vascular 20
Medicine, department of Nephrology 21
Albinusdreef 2, 2333 ZA, Leiden, The Netherlands 22
Inflammatory cells such as macrophages have been shown to increase heparanase activity by 351
activation of secreted pro-heparanase by cathepsin-L.(30, 31) While the absolute number of 352
macrophages remained equal between atrasentan treated and non-treated diabetic mice (F4/80 353
positive cells: 2.15 ± 0.37 vs. 2.53 ± 0.42 / glomerulus respectively), there was a shift from 354
pro-inflammatory M1 macrophages towards regulatory non-inflammatory CD206 positive 355
M2 macrophages in atrasentan treated mice (62.2 ± 11.1% vs. 44.8 ± 6.1%, p<0.01), resulting 356
in a similar distribution as was observed in non-diabetic apoE KO mice (64.8 ± 4.1 Figure 357
5A,B). Concomitant with this shift in macrophages’ phenotype and increased heparanase 358
expression, we also observed increased cathepsin-L protein expression in diabetic apoE KO 359
mice (27.3 ± 11.3% vs. 10.5 ± 2.8%, p<0.01 and a reduction by atrasentan (10.1 ± 5.1%, 360
Figure 5A,D). Notably, although cathepsin-L is more prominently present in tubular 361
epithelium, glomerular F4/80 positive macrophages also co-localize with cathepsin-L 362
expression (Supplementary Figure S2). 363
364
Atrasentan restores glycocalyx thickness on endothelial cells in a diabetic milieu by 365
reducing heparanase 366
To further study our hypothesis that atrasentan can reduce endothelial heparanase expression 367
under conditions of endothelial activation in diabetes, and subsequently can increase 368
glycocalyx thickness, we examined glycocalyx thickness on human umbilical vein 369
endothelial cells (HUVECs) in the presence of diabetic and control human serum. HUVECs 370
were cultured under flow (10 dyne/cm2) for 4 days, on top of a layer of human brain pericytes 371
(HBPs) to induce a quiescent endothelial phenotype and to resemble the in vivo cell-cell 372
interactions that determine this endothelial phenotype. Under control conditions, these cells 373
express a glycocalyx of 1.84 ± 0.36 µm as shown with the lectin wheat germ agglutinin 374
(WGA) (Figure 6). To mimic the conditions present in diabetes, we exposed the endothelial 375
Page 15 of 32 Diabetes
cells to serum of patients with poorly controlled diabetes. Importantly, while diabetes 376
obviously is characterised by hyperglycemia, plasma of diabetes patients contains a wide 377
range of factors that may cause endothelial activation, including advanced glycation end 378
products, chemokines such as MCP-1, and vasoactive peptides such as angiotensin and 379
endothelin (32). To mimic these circumstances, cells were incubated for 3 days with medium 380
supplemented with serum of poorly controlled diabetic patients and consequently, glycocalyx 381
thickness decreases to 1.12 ± 0.26 µm (p<0.05). Addition of 0.5 µM atrasentan to cells 382
cultured in the presence of diabetic serum restored glycocalyx thickness to 1.48 ± 0.19 µm 383
(p<0.05). The heparanase inhibitor OGT2115 also increased the glycocalyx thickness (1.38 ± 384
0.33 µm, p<0.05). Adding both compounds simultaneously, however, had no synergetic 385
effect (1,38 ± 0,33 µm, p<0.05, data not shown). Staining with the antibody 10E4, against the 386
N-acetylated and N-sulfated heparan sulfate domains, to look more closely at the specific 387
composition, showed similar results as the WGA staining (Figure 6A-B). 388
To further test the involvement of heparanase in modulation of the endothelial glycocalyx, we 389
analysed heparanase gene expression and heparanase protein presence at the luminal surface 390
of the endothelial cells (Figure 6C). In agreement with the in vivo studies, incubation with 391
diabetic serum for 3 days induced a 1.63 ± 0.27 fold increased luminal protein expression, 392
which was paralleled by an 1.46 ± 0.28 fold mRNA expression, compared with incubation of 393
non-diabetic serum (p<0.05). Supplementation of 0.5 µM atrasentan to these cells cultured in 394
the presence of diabetic serum, normalized both luminal heparanase protein expression, as 395
well as mRNA expression (to 1.19 ± 0.23 fold and 1.10 ± 0.11 fold, compared with control, 396
respectively). The heparanase inhibitor decreases luminal expression of heparanase 1.25 ± 397
0.22 fold, p<0.05, but not gene expression (1.2 ± 0.46 fold) and there was no amplification of 398
the effect of atrasentan. 399
400
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DISCUSSION 401
In this study, selective ETA receptor blockade in diabetic nephropathy is associated with 402
almost complete restoration of glomerular endothelial glycocalyx dimensions towards control 403
levels and reduction of albuminuria. Especially, the profound reduction of albuminuria occurs 404
in the absence of any changes in systemic blood pressure and metabolic activators, such as 405
high glucose levels. Both the in vivo data as well as the mechanistic studies in vitro show that 406
atrasentan is capable of reducing heparanase expression in the presence of a diabetic milieu. 407
This study provides a new mechanism of action for ongoing clinical studies with ETA 408
receptor blockers in diabetic nephropathy, where similar strong reductions in proteinuria were 409
observed in the presence of only minor hemodynamic effects (11). 410
411
There has been controversy both with respect to the mechanism of albuminuria, as well as the 412
possible consequences of albuminuria in diabetic nephropathy. Most experimental data point 413
to size selectivity of the glomerular filter. The glomerular glycocalyx, through its mesh of 414
glycosaminoglycans and associated proteins, constitutes a size selective hydrogel that covers 415
the surface and in particular the fenestrae (33). Disruption of this structure by enzymatic 416
treatment, or more recently by endothelial gene deletion of hyaluronan synthase 2, has been 417
shown to result in albuminuria (13, 34). Moreover, the high heparan sulfate content and 418
presence of sialated proteins may give the endothelial surface a net negative charge, thus 419
possibly further modulating the sieving of macromolecules. Since diabetes is associated with 420
endothelial dysfunction and reduced systemic glycocalyx dimensions (12, 15), restoration of 421
endothelial function and glycocalyx dimensions may thus result in prevention of albuminuria. 422
Such a therapy would be meaningful in the setting of diabetes where chronic exposure of 423
glomerular and tubular endothelium to glycated albumin has been shown to induce epithelial 424
inflammation and set the stage for tubulointerstitial disease (35). 425
426
Page 17 of 32 Diabetes
To corroborate the beneficial effects of atrasentan on endothelial function, we used 427
paramagnetic ferrous mononitrosyl-iron complex (MNIC) spin trap measurements: this 428
model allows for quantitative measurements of the amount of nitric oxide molecules 429
produced locally (27). Atrasentan increased nitric oxide production at the renal tissue level 430
(26), thus confirming endothelial ETB receptor stimulation and restoration of endothelial 431
function (36), despite the presence of diabetes. 432
433
To further address the mechanism behind the beneficial effects of atrasentan on heparanase 434
reduction and its effect on endothelial glycocalyx dimensions, we also studied the effect of 435
atrasentan on the endothelial glycocalyx in vitro. As the glycocalyx composition is critically 436
dependent upon shear, cellular environment and endothelial function, we used an 437
experimental set-up in which endothelial cells were exposed to laminar flow and cultured on 438
top of pericytes, to mimic as closely as possible the in vivo situation. Endothelial cells show a 439
remarkable heterogeneity throughout the vascular tree and may therefore differ in their 440
response to injury (37, 38). Despite this heterogeneity, HUVECs are capable to express 441
heparanase (39) and in this model, adding diabetic serum, thus mimicking the diabetic milieu, 442
increased endothelial heparanase expression. Heparanase is the main enzyme that can break 443
down heparan sulfate side-chains of glycosaminoglycans, and consequently glycocalyx 444
thickness was reduced. In line with our observations in mice, atrasentan reduced heparanase 445
expression through transcriptional regulation and restored the reduction of glycocalyx 446
thickness in the presence of diabetic serum. Atrasentan was as effective as a heparanase 447
inhibitor and the heparanase inhibitor did not amplify the effect of atrasentan, indicating that 448
direct modulation of endothelial heparanase expression may be a mechanism by which 449
atrasentan restores the glycocalyx. 450
451
Page 18 of 32Diabetes
Atrasentan has been studied previously in other diabetic animal models. In a streptozotocin 452
induced diabetic rat model atrasentan reduced the onset of albuminuria, independent of 453
changes in blood pressure (7, 40). Using the same model as in the present study, another ETA 454
selective blocker, Avosentan, was also shown to have strong anti-albuminuric effects (17). 455
Similar to our study, this was accompanied by anti-inflammatory effects, such as reduced 456
renal macrophages influx and additional decreased plasma levels of the inflammatory 457
markers MCP-1 and soluble ICAM-1. Such anti-inflammatory effects may have further 458
contributed to the reduction in heparanase expression that was observed in the diabetic mice, 459
as infiltrating monocytes have been shown to contribute to activation of secreted pro-460
heparanase (41). This is further supported by our observations that atrasentan reduced 461
glomerular cathepsin-L expression, the enzyme that activates pro-heparanase; cathepsin-L 462
expression co-localized with inflammatory glomerular macrophages. 463
464
Another ETA receptor blocker, sitaxsentan, was shown to reduce podocyte loss in ADR-465
induced nephropathy(42). However, in our model, we did not observe a change in podocyte 466
numbers. Furthermore, we did not see changes in systemic blood pressure during atrasentan 467
treatment. However, a reduction in glomerular capillary pressure cannot be ruled out as 468
possible mechanism to explain the beneficial effects on glomerular ultrastructure and 469
glomerular endothelial glycocalyx function. Particularly as micropuncture studies in rats have 470
demonstrated the presence of increased glomerular capillary pressure in STZ diabetes models 471
(43). Unfortunately, this technology cannot be applied to mice. 472
473
While our model only studied the short term effects of atrasentan in already developed 474
diabetic nephropathy, it would of course be relevant to know whether prolonged restoration 475
of the glomerular glycocalyx also results in restoration of the cellular morphology or 476
Page 19 of 32 Diabetes
prevention of (further) renal lesions. Both the effectiveness in prevention of albuminuria as 477
well as the fact that the glomerular glycocalyx functions as a molecular scaffold that 478
modulates renal inflammation makes this question pertinent. Unfortunately, the long duration 479
of the model, which is required to faithfully replicate changes seen in human diabetic 480
nephropathy, precluded such follow up studies in STZ treated animals. This does, however, 481
not detract from the fact that the current study not only corroborates the rationale for clinical 482
use of ETA selective receptor blockade in diabetic nephropathy; given the systemic nature of 483
loss of glycocalyx in diabetes, it also provides a mechanism of action which can be monitored 484
non-invasively (44) in patients before and during treatment. 485
486
ACKNOWLEDGEMENTS 487
We thank Prof E. Bouwman (Inorganic Chemistry, Leiden University) for the use of the 488
electron paramagnetic resonance facilities. 489
An abstract containing data from this study was presented at the American Society of 490
Nephrology Kidney Week 2014, November 11-16, 2014, Philadelphia, PA. 491
This study was supported by the Glycoren consortium grant of the Dutch Kidney Foundation 492
(CP09.03) and an AbbVie study grant (REN-11-0026). 493
No potential conflicts of interest relevant to this article were reported. 494
495
AUTHOR CONTRIBUTIONS 496
M.B. designed experiments, researched and analysed data, and wrote and revised the 497
manuscript. M.A., A.K., M.D., D.L. and E.F. helped to acquire and interpret data and 498
critically revised the manuscript. J.V., A.K, A.Z. and H.G. critically revised the manuscript 499
for important intellectual content. B.B. and T.R. conceived, designed and supervised the 500
study and critically revised the manuscript. T.R. is the guarantors of this work and, as such, 501
Page 20 of 32Diabetes
had full access to all the data in the study and take responsibility for the integrity of the data 502
and the accuracy of the data analysis. 503
504
Page 21 of 32 Diabetes
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The diabetic apoE KO mouse model recapitulates the features of human diabetic nephropathy. A) Healthy glomerulus of non-diabetic apoE KO. Heterogeneous lesions in age-matched apoE KO mice 14 weeks
after induction of diabetes with STZ show mesangial expansion (B) and mesangiolysis (C,D) and
subsequently glomerular hypertrophy, as quantified in (E) and (F). Transmission electron microscopic image (TEM, D) reveals a typical pathological process of mesangial foam cell formation and increased extracellular matrix deposition, resulting in decreased endothelial and mesangial cell interaction. TEM microscopy shows differences in cationic ferritin coverage between non-diabetic (G) and diabetic apoE KO mice (H). Also the occurrence of podocyte foot processes effacement can be observed in (H). Data are shown as mean ± SD, *P<0.05, n = 8. Scale bars: 20 µm (A-C); 5 µm (D); 500 nm (G-H). ApoE = apoE KO mice; DM = diabetic
apoE KO mice; DM + A = diabetic apoE KO mice + atrasentan. 175x359mm (300 x 300 DPI)
Page 25 of 32 Diabetes
Atrasentan reduces albuminuria in diabetic apoE KO mice. A) Changes in urinary albumin-creatinine ratios (ACR) from baseline to 4 weeks after treatment, as indicated by percent from baseline. Data are shown as mean ± SD, n = 19-23 (8 for SBP), *P<0.01. B-C) Glomerular morphology is not affected by
treatment with atrasentan (DM + A) for 4 weeks, compared to diabetic mice (DM). After treatment, no change in blood glucose levels (D) and systolic blood pressure (E, n = 8) is observed. ApoE = ApoE KO
mice, scale bars: 20 µm. 199x350mm (300 x 300 DPI)
Page 26 of 32Diabetes
Atrasentan restores endothelial glycocalyx coverage. A-B) Representative TEM microscopic images of cationic ferritin bound to the negatively charged endothelial glycocalyx in glomeruli of diabetic (A) and
atrasentan treated diabetic (B) mice. C) Quantification of endothelial cationic ferritin coverage in capillary
loops in 3 glomeruli of 3 mice, shown as mean percentage of total capillary length ± SD. D) Confocal fluorescent image of a glomerular capillary loop, stained for endothelial cells (CD31, red) and luminal glycocalyx (fluorescent-labeled lectin Lycopersicon esculentum, LEA, green). Arrow: line of interest for
intensity plot E) Example of fluorescence intensity plot, depicting the area used for quantification of luminal glycocalyx thickness, which is determined by the distance of the CD31 peak to the half maximum intensity
of the LEA peak. F) Quantification of LEA thickness in capillary loops in 3 glomeruli of 3 mice, shown as mean ± SD. Scale bars: 500 nm (A-B), 5 µm (D). *P<0.05 compared with ApoE and DM + A. ApoE = ApoE
KO mice, DM = diabetic apoE KO mice, DM + A = diabetic apoE KO mice + atrasentan. 120x82mm (300 x 300 DPI)
Page 27 of 32 Diabetes
Atrasentan increases nitric oxide bioavailability. A) Example of electron paramagnetic resonance (EPR) spectrum of frozen murine diabetic kidney sample after atrasentan treatment. The characteristic triplet
structure of mononitrosyl-iron complex (MNIC, arrow) represents the formation of local nitric oxide (NO). B) Quantification of renal NO formation, shown as mean MNIC ± SD, n = 8-9. *P<0.05, compared with DM.
ApoE = ApoE KO mice, DM = diabetic apoE KO mice, DM + A = diabetic apoE KO mice + atrasentan. 95x107mm (300 x 300 DPI)
Page 28 of 32Diabetes
Atrasentan changes glomerular M1 to M2 macrophage ratio and reduces heparanase and
cathepsin-L expression. A) Representative fluorescent images of glomerular F4/80 positive (arrowhead) and F4/80-CD206 double positive macrophages (arrow, top row), heparanase (HPSE) expression (middle
row) and cathepsin-L (CTSL) expression (bottom row) in ApoE KO mice (ApoE), non-treated diabetic apoE KO mice (DM) and diabetic apoE KO mice treated with atrasentan (DM + A), scale bar: 20 µm. B-D)
Quantification shown as mean ± SD, *P<0.01, compared with ApoE and DM + A, n = 8. 136x103mm (300 x 300 DPI)
Page 29 of 32 Diabetes
Atrasentan restores glycocalyx thickness on HUVEC. A) Top: schematic drawings showing the area of interest for quantification (dotted line). Bottom: confocal fluorescent Z-axis average-intensity projections of human umbilical cord endothelial cells (HUVEC) cultured on top of human brain pericytes under laminar flow
for 4 days. Left: Wheat germ-agglutinin (WGA, red) lectin and specific anti-heparan sulfate (10E4, green) staining; Right: anti-heparanase (HPSE) staining. B) Glycocalyx thickness is quantified by estimating the
distance from the half maximum signal of the nuclear staining to the half maximum signal at the luminal end of WGA and 10E4 staining. C) Endothelial HPSE protein and mRNA expression are shown as relative to NHS. Protein expression is quantified as average intensity staining in the area of interest (A). Data are shown as mean ± SD, *P<0.05, compared with NHS; #P<0.05, versus each treatment, †P<0.05, versus atrasentan
treatment, n = 4-5. Scale bars: 10 µm. NHS = normal human serum (control), DHS = diabetic human serum, DHS + A = DHS + 5 µM Atrasentan, DHS + O = DHS + heparanase inhibitor (OGT2115).
120x82mm (300 x 300 DPI)
Page 30 of 32Diabetes
SUPPLEMENTARY DATA
Supplementary figure S1. Diabetes increases endothelial heparanase mRNA expression. Quantification of glomerular endothelial heparanase mRNA expression, relatively to non-diabetic
apoE KO mice (ApoE). Murine heparanase was identified with forward 5’-
GAGCGGAGCAAACTCCGAGTGTATC-3’ and reverse 5’-GATCCAGAATTTGACCGTTC
AGTTGG-3’ primers. Data is shown as mean ± SD, n = 3-4, *P<0.05, compared with ApoE. DM =
diabetic apoE KO mice, DM + A = diabetic apoE KO mice + atrasentan.
Page 31 of 32 Diabetes
SUPPLEMENTARY DATA
Supplementary figure S2. Glomerular macrophages co-localize with cathepsin-L. Representative
fluorescent images of glomerular F4/80 positive macrophages (red) and cathepsin-L (green) in
diabetic apoE KO mouse. Arrows indicate co-localization in merged image, scale bar: 20 µm.