Long-Term Endothelin-A Receptor Antagonism Provides Robust ... · marks of the SCD milieu, including hypoxia, oxidative stress, and thrombosis.17 This theoretic connection between

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Long-Term Endothelin-A Receptor AntagonismProvides Robust Renal Protection in Humanized SickleCell Disease Mice

Malgorzata Kasztan,* Brandon M. Fox,* Joshua S. Speed,* Carmen De Miguel,*Eman Y. Gohar,* Tim M. Townes,† Abdullah Kutlar,‡ Jennifer S. Pollock,*§ andDavid M. Pollock*§

*Cardio-Renal Physiology and Medicine, Department of Medicine, and †Department of Biochemistry and MolecularGenetics, University of Alabama at Birmingham, Birmingham, Alabama; and ‡Division of Hematology and Oncology,and §Department of Medicine, Medical College of Georgia, Augusta University, Augusta, Georgia

ABSTRACTSickle cell disease (SCD)–associated nephropathy is a major source of morbidity and mortality in patientsbecauseof the lackof efficacious treatments targeting renalmanifestationsof thedisease.Here,wedescribealong-term treatment strategy with the selective endothelin-A receptor (ETA) antagonist, ambrisentan,designed to interfere with the development of nephropathy in a humanized mouse model of SCD.Ambrisentan preserved GFR at the level of nondisease controls and prevented the development ofproteinuria, albuminuria, and nephrinuria.Microscopy studies demonstrated prevention of podocyte lossand structural alterations, the absence of vascular congestion, and attenuation of glomerulosclerosis intreated mice. Studies in isolated glomeruli showed that treatment reduced inflammation and oxidativestress. At the level of renal tubules, ambrisentan treatment prevented the increased excretion of urinarytubular injury biomarkers. Additionally, the treatment strategy prevented tubular brush border loss, di-minished tubular iron deposition, blocked the development of interstitial fibrosis, and prevented immunecell infiltration. Furthermore, the prevention of albuminuria in treated mice was associated with preser-vation of corticalmegalin expression. In a separate series of identical experiments, combined ETA and ETBreceptor antagonism provided only some of the protection observed with ambrisentan, highlighting theimportance of exclusively targeting the ETA receptor in SCD. Our results demonstrate that ambrisentantreatment provides robust protection from diverse renal pathologies in SCDmice, and suggest that long-term ETA receptor antagonism may provide a strategy for the prevention of renal complications of SCD.

J Am Soc Nephrol 28: 2443–2458, 2017. doi: https://doi.org/10.1681/ASN.2016070711

Sickle cell disease (SCD) is an autosomal recessivemonogenetic blood disorder that affects approxi-mately 100,000 people in the United States and mil-lions globally.1,2 The central pathology of the diseaseis the polymerization of mutant hemoglobin S (HbS).This process sets in motion a cascade of events, begin-ning with erythrocyte sickling, endothelial activation,and vaso-occlusion, that ultimately result in chronictissue hypoxia, inflammation, and organ damage.3 Ad-vances in themanagement of SCD throughout the pasthalf-century have led to a progressive increase in lifeexpectancy with a concomitant increase in the preva-lence of chronic disease manifestations.4,5 Sickle cell

nephropathy now represents one of the most seri-ous organ-specific complications facing patientswith SCD. Renal pathologies in SCD occur at

Received July 1, 2016. Accepted February 13, 2017.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Prof. David M. Pollock, Cardio-Renal Physiol-ogy and Medicine, Department of Medicine, Division of Ne-phrology, University of Alabama at Birmingham, Birmingham, AL35233. Email: [email protected]

Copyright © 2017 by the American Society of Nephrology

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both the structural and functional level and include deterio-ration of the glomerular filtration barrier, progressive loss offunctional glomeruli, tubulointerstitial injury, defectiveurine-concentrating ability, and dysfunction of the distalnephron.6 Evidence of nephropathy, as manifested by albu-minuria or proteinuria, occurs in nearly two-third of adultswith SCD, and presently, almost one in five patients with SCDdie of renal disease.7,8 This level of disease burden occurs dueto lack of availability of robustly renal-protective therapiestargeted to the mechanisms that underlie sickle nephropathy.

Endothelin-1 (ET-1) is a signaling peptide produced by di-verse cell types that exerts its physiologic and pathophysiologicactions by binding to two receptor subtypes, ETA and ETB.9,10

ETA receptor activation induces vasoconstriction, inflamma-tion, mitogenesis, and nociception.11–15 These effects can becounteracted by ETB activation in some tissues, highlightingthe importance of the balance between the two receptorsubtypes.16 ET-1 expression is induced by established hall-marks of the SCD milieu, including hypoxia, oxidative stress,and thrombosis.17 This theoretic connection between SCDand ET-1 overproduction was validated by multiple studiesthat demonstrated elevated ET-1 levels in both plasma andurine of patients with SCD.17 In the kidney, ET-1 has beenimplicated as a mechanistic contributor to development ofproteinuria in various forms of CKD.18–22 Moreover, the ele-vated urinary ET-1 excretion reported in patients with SCDcorrelates with microalbuminuria,23 suggesting a pathophys-iologic link between ET-1 induction and renal injury in SCD.Direct evidence to demonstrate that ET-1 contributes to renaldysfunction in SCD comes from a study by Sabaa and col-leagues who reported that SCD mice treated with the dualET receptor antagonist, bosentan, were protected from hypoxia-induced decreases in renal blood flow and renal inflammation.24

These studies have provided both clinical and preclinicalevidence to support the hypothesis that ET-1 signaling is amechanistic mediator of organ-specific pathology in SCD.However, no study has provided rigorous evidence to charac-terize ET receptor antagonism as a treatment strategy for renalcomplications of SCD.

This study aimed to investigate the renal protectivepotentialof ETreceptor antagonism in the treatment of sickle nephrop-athy and determine the importance of receptor subtype target-ing and duration of treatment.We demonstrate that long-termETA receptor antagonism can prevent the development ofmany of the glomerular and tubular pathologies that charac-terize the nephropathy associated with SCD. Additionally,short-term ETA receptor antagonism demonstrated the abilityto reduce, but not fully reverse, the severity of establishednephropathy. Dual ETA/B receptor antagonism was examinedunder the same duration treatment protocols and was foundto provide less renal protection than ETA receptor antagonismalone. These results establish ET receptor antagonism as ahighly efficacious treatment strategy for sickle nephropathyand highlight that maximal benefit is provided by long-termantagonism of the ETA receptor.

RESULTS

Characteristics of Experimental AnimalsVehicle-treated 14-week-old HbSS mice had significantlyhigher spleen-to–body weight ratio and daily urine volume,and decreased urine osmolality, when compared with age-matched vehicle-treated HbAA mice. Two-week treatmentwith selective ETA receptor antagonist, ambrisentan, signifi-cantly reduced spleen-to–body weight ratio of HbSS mice. Noparameters were changed with nonselective ETA/B receptorantagonist, A-182086, treatment in HbSS mice (Tables 1 and2). Neither antagonist had any effect on the characteristics ofcontrol mice (Tables 1 and 2). HbSS mice had elevated plasmaET-1 concentration compared with controls, and both 2-weekand 10-week ambrisentan treatment protocols significantlyreduced plasma ET-1 level (Figure 1A, Table 3). This resultsuggests that in SCD the ETA receptor signals through a pos-itive feedback loop to increase ET-1 expression.

Short-Term ET Receptor Antagonism AttenuatesGlomerular and Tubular InjuryTo investigate the contribution of ET signaling to the mainte-nance and progression of established nephropathy in SCD, wefirst examined the effect of 2-week treatment with ETreceptorantagonists on glomerular and tubular dysfunction in HbSSand HbAA mice. Vehicle-treated HbSS mice had significantlyhigher proteinuria, albuminuria, nephrinuria, and glomerularpermeability to albumin (Palb) when compared with HbAAcontrols (Table 3). Ambrisentan significantly reduced albu-minuria, nephrinuria, and Palb in HbSS mice, whereasA-182086 significantly attenuated only Palb, and the effect ofdual antagonism did not reach statistical significance in thereduction of albuminuria and nephrinuria (Table 3). Theseresults suggest that ETA activation contributes to the mainte-nance of dysfunction of the glomerular filtration barrier inestablished sickle nephropathy, a finding that is consistentwith previous results from our laboratory.22

We also determined if ETsignaling contributes to themain-tenance of tubular injury in SCD, because final urinary albu-min excretion is the result of both glomerular filtration andproximal tubular uptake. Thus, we measured urinary excre-tion of kidney injury marker 1 (KIM-1) and N-acetyl-b-D-glucosaminidase (NAG), markers of proximal tubule injury.Both markers were significantly increased in vehicle-treatedHbSS mice compared with controls. Ambrisentan signifi-cantly attenuated KIM-1 and NAG, whereas A-182086 hadno significant effect on these markers in HbSS mice (Table 3).Neither antagonist had any effect on markers of glomerular ortubular injury in control HbAA mice (Table 3).

Long-Term Ambrisentan Treatment Prevents Onset ofGlomerular InjuryTo investigate the pathophysiologic relevance of ETsignaling inthe development of sickle nephropathy, we examined the effectof 10-week treatment (fromweaning into adulthood) with ET

2444 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2443–2458, 2017


receptor antagonists on proteinuria in HbSS mice. SelectiveETA receptor antagonist prevented proteinuria in HbSS micewhen compared with vehicle-treated mice (1.160.3 versus3.5760.8 mg/24 h, respectively), whereas the nonselectiveETA/B receptor antagonist had no effect on urinary proteinexcretion (Figure 1B).

Next, we examined the role of ET signaling in the develop-ment of sickle cell glomerulopathybymeasuring structural andfunctional markers of glomerular injury in HbSS mice. Bothambrisentan and A-182086 prevented the increase in Palb inglomeruli isolated from HbSS mice (Figure 1C). Moreover,both antagonists prevented increases in nephrinuria (Figure1E). However, only the selective ETA receptor antagonistprevented albuminuria in HbSS mice (Figure 1D). Impor-tantly, ambrisentan treatment preserved GFR to the level ofnondisease controls (Figure 1F), whereas A-182086 treat-ment had no effect on the decline in GFR observed inHbSS mice. (Figure 1F). Histologic analysis of glomerularstructure showed glomerular hypertrophy, basement mem-brane of Bowman’s capsule thickening, glomerulosclerosis,and vascular congestion in vehicle-treated HbSS mice. Only

ambrisentan significantly prevented or attenuated all of thesechanges in glomerular morphology in HbSS mice (Figure 2).Neither antagonist had any effect on markers of glomerularstructure or function in control HbAA mice (SupplementalFigures 1 and 2). These results suggest that ETA activationcontributes to the development and progression of glomer-ulopathy in SCD.

Long-Term Ambrisentan Treatment Prevents Onset ofPodocyte InjuryPodocyte injury is a key feature of alteration in the integrity ofthe glomerular filtration barrier and, ultimately, proteinuricnephropathy.Thus,we examined the effect of the long-termETreceptor antagonism on podocyte phenotypic changes. Podo-cyte number per glomerulus, as determined byWilms tumor 1(WT-1) immunohistochemistry,was significantly decreased invehicle-treatedHbSSmice, andwas concomitant with reducedmRNA levels of the podocyte markers nephrin, podocin, andsynaptopodin.Both antagonists preventedpodocyte loss, dem-onstrated by WT-1–positive glomerular cells and mRNA ex-pression of podocyte markers (Figure 3, A–F). Transmission

Table 1. Characteristics of experimental animals after 2 weeks of treatment with ET receptor antagonists

CharacteristicsHbAA HbSS

Vehicle Ambrisentan A-182086 Vehicle Ambrisentan A-182086

Body weight, g M: 25.761.2 M: 28.860.8 M: 26.860.4 M: 27.561.1 M: 27.560.8 M: 28.260.5F: 22.361.0 F: 21.760.5 F: 21.760.7 F: 21.961.0 F: 21.160.9 F: 23.060.4

Right kidney/body weight, mg/g 6.660.3 6.560.4 6.360.4 7.460.4 7.660.5 7.860.5Left kidney/body weight, mg/g 6.260.3 5.860.3 5.960.3 7.260.4 7.760.5 7.060.4Spleen/body weight ratio, % 0.960.1 0.860.1 0.960.1 6.960.3a 5.760.3a,b 7.060.5a

Water intake, ml/24 h 3.260.3 4.760.5 4.660.6 5.260.5 6.060.5 5.860.5Urine excretion, ml/24 h 1.060.2 1.360.1 1.660.3 2.160.3a 2.360.4a 2.760.3a

Urine osmolality, mOsm/kg 24826147 22996121 26016121 11776135a 15516174a 1624680a

Systolic BP, mmHg 12061 11962 12161 12361 11963 12262Serum creatinine, mg/dl 0.0960.01 0.1060.01 0.1260.01 0.1160.01 0.1160.01 0.0960.01

Data are means6SEM (n=12–14 in HbSS groups and n=6–9 in HbAA groups). M, male; F, female.aP,0.05 versus HbAA vehicle.bP,0.05 versus HbSS vehicle.

Table 2. Characteristics of experimental animals after 10 weeks of treatment with ET receptor antagonists

CharacteristicsHbAA HbSS


Body weight, g M: 32.260.9 M: 30.560.9 M: 32.161.3 M: 29.360.4 M: 28.660.5 M: 27.561.1F: 24.961.5 F: 24.360.7 F: 22.260.7 F: 24.260.7 F: 25.760.8 F: 24.260.7

Right kidney/body weight, mg/g 6.660.3 6.460.3 6.360.2 6.860.3 6.560.3 6.460.3Left kidney/body weight, mg/g 5.660.5 5.860.2 6.060.1 6.460.2 6.060.2 6.060.2Spleen/body weight ratio, % 0.860.1 1.060.1 0.860.1 7.060.3a 5.960.4a 6.760.4a

Water intake, ml/24 h 3.860.6 4.060.3 3.560.4 4.660.4 6.361.2 6.060.3Urine excretion, ml/24 h 1.060.3 0.960.1 1.060.2 2.060.3a 2.160.4a 1.860.1a

Urine osmolality, mOsm/kg 25746415 22576185 23366247 1519642a 15196136a 13706149a

Systolic BP, mmHg 12162 11562 12164 12564 11864 12663Serum creatinine, mg/dl 0.1060.01 0.1160.01 0.1060.01 0.0960.01 0.1060.01 0.1060.01

Data are means6SEM (n=9–10 in HbSS groups and n=6–9 in HbAA groups). M, male; F, female.aP,0.05 versus HbAA vehicle.bP,0.05 versus HbSS vehicle.

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electron microscopy studies in glomeruli of vehicle-treatedHbSS mice showed evidence of more podocyte foot processfusion and effacement, and glomerular basement membrane

thickening, whereas prevalence of thesestructural defects was similar to vehicle-treated nondisease controls in mice treatedwith both antagonists (Figure 3G). Neitherantagonist had any effect on markers ofpodocyte injury in control HbAA mice(Supplemental Figure 3). These resultssuggest that ETA activation contributes tothe development and progression of podo-cyte injury in SCD.

Long-Term Ambrisentan TreatmentPrevents Glomerular DysfunctionTo elucidate themechanism underlying thenephroprotective effect of ET receptor an-tagonism on glomeruli of HbSS mice, weexamined endothelial activation, inflam-mation, and oxidative stress, hallmarks ofglomerular dysfunction. Isolated glomerulifrom HbSS mice treated with both antago-nists demonstrated nondisease control–level sensitivity to phorbol 12-myristate13-acetate–stimulated reactive oxygenspecies (ROS) production (Figure 4G). Bycontrast, only the selective ETA receptorantagonist prevented the induction of vas-cular adhesion molecules and proinflam-matory marker expression in glomerulifrom HbSS mice (Figure 4, D–F). Neitherantagonist had any effect on glomerulardysfunction in control HbAA mice (Sup-plemental Figure 4). These results suggestthat ETA receptor antagonism successfullyprotects from ET-mediated glomerular al-terations in sickle nephropathy.

Long-Term Ambrisentan TreatmentPrevented Tubular InjuryOn the basis of data from the short-termtreatment protocol, we analyzedmarkers ofinjury and histologic structure of the prox-imal tubules in HbSS mice. Increasedurinary KIM-1 and NAG excretion wereprevented by ambrisentan compared withvehicle-treated HbSS mice (Figure 5, A andB). Moreover, ambrisentan prevented renalinflammation and fibrosis, possiblythrough blocking T cell and macrophageinfiltration (Figure 6, A–F) as well as theinduction of proinflammatory mediators(Figure 6, G–I). In contrast, antagonismof both ET receptors had no significant

effect on tubular injury in HbSS mice when compared withthe selective ETA receptor antagonist (Figures 5 and 6). Theseresults were confirmed by histologic analysis where

Figure 1. Long-term selective ETA receptor antagonism preserves glomerular function bypreventing glomerular injury in humanized sickle cell mice. Plasma ET-1 and markers ofrenal function from genetic control (HbAA) and humanized sickle mice (HbSS) treated withvehicle, the selective ETA antagonist, ambrisentan, or the combined ETA/B antagonist,A-182086, for 10 weeks beginning at 4 weeks of age. (A) Depicts the average ET-1 inplasma after 10-week treatment protocol. (B) Depicts the average proteinuria after 10-week treatment protocol. (C) Depicts the average Palb after 10-week treatment protocol.(D) Depicts the average urinary albumin excretion after 10-week treatment protocol. (E)Depicts the average urinary nephrin excretion after 10-week treatment protocol. (F) De-picts the average GFR after 10-week treatment protocol. Data are mean6SEM; *P,0.05versus vehicle-treated HbAA; #P,0.05 versus vehicle-treated HbSS; n=9–10 in HbSS andn=9 in vehicle-treated HbAA group.






ambrisentan preserved brush border thickness (Figure 5, Dand G), prevented interstitial fibrosis (Figure 5, E and H), andreduced tubular iron deposition associated with nondiseaselevels of urinary neutrophil gelatinase-associated lipocalin(NGAL), iron binding protein, excretion inHbSSmice (Figure5, C, F and I). Neither antagonist had any effect on tubularstructure and function in control HbAA mice (SupplementalFigures 5 and 6).

Long-Term ET Receptor Antagonism TreatmentPreserves Tubular Megalin ExpressionThe contradictory observations that treatment of HbSS micewith either selective or nonselective ET antagonists preventedthe increase in Palb, whereas only the selective ETA antagonistprevented albuminuria, led us to investigate if the two antag-onists had differing effects on proximal tubular protein han-dling. We measured expression of the receptors involved inproximal tubular endocytosis of filtered albumin. HbSS micehad significantly lower megalin mRNA and protein expressionwhen compared with HbAA mice, and ambrisentan, but notA-182086, restored expression to nondisease levels (Figure 7,A–C). In addition, suppressed neonatal Fc receptor (FcRn)mRNA expression was observed only in ETA/B antagonist–treated HbSS mice (Figure 7F). Neither antagonist had anyeffect on proximal tubule protein handling receptors in con-trol HbAA mice (Supplemental Figure 7). These results sug-gest that proximal tubular ETB receptor signaling may play arole in albumin uptake regulation and contribute to the degreeof albuminuria in HbSS mice.

DISCUSSION

Renal complications of SCD include a variety of glomerularand tubular abnormalities, however, the pathogenesis of thesecomplications remains unclear. This study demonstrated thatlong-term ETA receptor antagonism prevented the developmentand progression of sickle nephropathy, thus demonstrating thatETA receptor signaling is a critical mechanistic mediator inrenal complications associated with SCD. These studies pro-vide the basis for the use of ETA receptor antagonism as a

potential therapeutic approach for the prevention of renalinvolvement in SCD.

Abundant evidence implicates endothelial dysfunction inmany proteinuric renal diseases,19,20,22,25–27 suggesting thatactivation of ET receptors may lead to glomerular endothelialand tubular dysfunction, thereby contributing to proteinuria.Our group has previously demonstrated that hypoxia in-creases glomerular ET-1 expression in C57BL/6J mice, butnot in endothelial-specific ET-1 knockout mice.28 Also, ET-1mRNA expression in glomeruli isolated from HbSS mice wasshown to be significantly elevated.22 These data may suggest amechanism for ET-1–induced nephropathy in SCD. In thisstudy, we observed significantly elevated plasma levels ofET-1 and proteinuria in HbSS mice, consistent with priorreports of elevated ET-1 levels and proteinuria in patientswith SCD.23,29–31 Albuminuria is linked with progression ofnephropathy in SCD32,33 and has been associated with a de-cline in GFR and glomerular permselectivity in SCD.34 This isconsistent with other proteinuric renal diseases where pro-teinuria correlates with decline in GFR.35 In line with humandisease, our results in humanized SCDmice provided evidencethat prevention of proteinuria, with selective ETA receptorantagonism, was associated with preserved GFR.

Our observations suggest a pathophysiologic link betweenincreasedETsignaling and the impairment of glomerular func-tion in SCD.Moreover, given the often opposing actions of ETAand ETB receptors, we asked the important question ofwhether selective ETA or nonselective ETA/B receptor antago-nism provides superior or equivalent renal protection in SCD.This is particularly relevant given that both the combinedETA/B receptor antagonists, bosentan and macitentan, andthe selective ETA receptor antagonist, ambrisentan, are cur-rently Food andDrug Administration–approved for treatmentof pulmonary arterial hypertension. The clinical experiencethat now exists with both selective and nonselective ET recep-tor antagonists minimizes barriers in the path to clinical trans-lation of our findings.

The contribution of ETB receptor signaling to proteinuriaand Palb in SCD could not be directly assessed using a selectiveETB receptor antagonist in vivo because of hypertension, re-duced ET-1 clearance, and increased ETA receptor activation

Table 3. Short-term ET receptor antagonism attenuates glomerular and tubular injury in HbSS mice

VariablesHbAA HbSS


Plasma ET-1, pg/ml 0.660.1 0.860.2 0.760.1 1.860.2a 1.060.1b 1.660.2a

Urinary protein excretion, mg/24 h 1.860.3 1.760.5 2.160.6 3.260.4a 2.460.3b 3.560.4a

Palb 0.1260.02 0.1360.02 0.1260.02 0.4860.05a 0.2460.03b 0.2760.04a,b

Urinary albumin excretion, mg/24 h 30.167.9 25.665.7 26.067.7 81.7618.0a 35.266.2b 108.3627.1a

Urinary nephrin excretion, ng/24 h 7.561.9 5.561.8 10.864.1 52.867.6a 31.662.1a,b 37.863.1a

Urinary KIM-1 excretion, pg/24 h 3768 112626 113649 272646a 122622b 323634a

Urinary NAG excretion, mU/24 h 9.461.6 5.462.7 6.462.8 40.268.3a 15.762.9b 27.567.1a

Data are means6SEM (n=12–14 in HbSS groups and n=6–9 in HbAA groups).aP,0.05 versus HbAA vehicle.bP,0.05 versus HbSS vehicle.






Figure 2. Histological analysis reveals that selective ETA receptor antagonism preserves glomerular morphology and prevents vascular congestionin humanized sickle cell mice. Histologic examination of the renal cortex of genetic control (HbAA) and humanized sickle mice (HbSS) treated withvehicle, the selective ETA antagonist, ambrisentan, or the combined ETA/B antagonist, A-182086, for 10 weeks beginning at 4 weeks of age. (A)Depicts representative Masson trichrome–stained sections of glomeruli. Original magnification, 340 (scale bar=50 mm). (B) Depicts quantificationof (A) represented as sclerosis index score. (C) Depicts glomerulomegaly represented as mean area of glomeruli (square micrometer). (D) Depictsnumber of glomeruli per square millimeter. (E) Depicts representative hematoxylin and eosin–stained sections of glomeruli. Original magnification,340 (scale bar=50 mm). (F) Depicts glomerular congestion represented as percentage of glomeruli with congestion. (G) Depicts basementmembrane of Bowman’s capsule thickening score. Data are mean6SEM; n=5 in HbAA and HbSS groups; *P,0.05 versus vehicle-treated HbAA;#P,0.05 versus vehicle-treated HbSS. All of the glomerular characteristics were counted in ten sections per slide (minimum 20 glomeruli).



Figure 3. ETA receptor antagonism prevents podocyte loss and preserves podocyte structure in humanized sickle cell mice. Immu-nohistochemical examination and mRNA expression of markers of podocyte injury of kidneys from genetic control (HbAA) andhumanized sickle mice (HbSS) treated with vehicle, the selective ETA antagonist, ambrisentan, or the combined ETA/B antagonist,A-182086, for 10 weeks beginning at 4 weeks of age. (A) Depicts representative WT-1–positive stained sections of glomeruli. Original



that result from ETB receptor antagonism.36 Our results dem-onstrated that treatment with a selective ETA receptor antag-onist significantly attenuated Palb in glomeruli from HbSSmice. Treatment with the combined ETA/B receptor antagonisthad a similar effect, indicating that the effect of ET-1 on Palb ismost likely exclusively mediated by ETA receptors. On thebasis of the lower expression of ETB receptors comparedwith ETA receptors in glomeruli,22,37 our results support aspecific role of ETA receptors in controlling Palb and are con-sistent with previous studies that have shown that ETB recep-tor antagonism does not alter ET-1–induced elevation of Palbin vitro.18 However, because Palb measurements are performedin isolated decapsulated glomeruli independent of hemody-namic influences, a potential role of ETB receptors on glomer-ular filtration through hemodynamic mechanisms in SCDcannot be excluded. The mechanism contributing to glomer-ular injury in mice treated with the combined ETA/B receptorantagonist may be an inability to prevent congestion in glo-merular capillaries, ultimately leading to impairment of renalhemodynamics evidenced by reduced GFR. This idea is inaccordance with other studies showing a relationship betweenearly alterations in renal hemodynamics and heightened his-tologic injury after renal ischemia in SCD.38 Moreover, ratslacking ETB receptors develop more severe renal injury andproteinuria in response to hyperglycemia39 or nephrectomy.40

In contrast, we observed significant decreases in urinary albu-min and protein excretion only in HbSS mice treated with theselective ETA receptor antagonist, again suggesting a crucialrole of ETA receptors in mediating glomerular injury in SCD.

Intact podocytes are essential for the integrity of theglomerular filtration barrier and podocyte injury leads toalbuminuria.41,42 Importantly, podocytes express ETA andETB receptors suggesting a target for elevated ET-1. In addi-tion, several studies have shown that disruption of the podo-cyte actin cytoskeleton occurs after exposure to ET-1.20,27

Moreover, previous studies have shown a direct effect ofchronic ET-1 infusion on urinary excretion of nephrin, amarker of podocyte injury.18 Interestingly, 1-week adminis-tration of a selective ETA receptor antagonist attenuated thiseffect in SCD mice, suggesting that antagonism of ETA recep-tors may prevent podocyte injury in SCD.22 Here, we provideevidence that ET-1, via ETA receptor activation, mediates po-docyte injury in SCD, and this effect can be prevented bychronic ETA receptor antagonism. Other investigators pro-vided similar evidence in diabetic and nondiabetic glomerularinjury18,43–45 and preeclampsia.46 When considering our

findings in the context of these prior studies, the data suggestthat disruption of glomerular structure by ET-1, via ETAreceptor activation, leads to alterations in podocyte morphol-ogy and function, resulting in increased Palb and albuminuria/proteinuria in SCDmice. However, a recently published studydemonstrated beneficial effects of podocyte-specific deletionof both ETA and ETB receptors, resulting in protection frompodocyte loss and mesangial matrix expansion in diabetic ne-phropathy.25 Further studies with the use of podocyte-specificETA or ETB receptor knockout mice are required to resolve thisimportant question in SCD.

In this study, a protective effect on glomerular congestionand size was observed in HbSS mice treated with the selectiveETA, but not the dual ETA/B, receptor antagonist. In contrast,Lenoir et al. reported significant benefit on glomerular struc-ture in hypoxia-exposed SCD mice treated with the dual ETreceptor antagonist, bosentan.47 However, bosentan is 30times more selective for ETA versus ETB receptors,48 andwhen considered in the context of this study, this suggeststhat renal protection in previous studies may have been me-diated primarily by blockade of ETA receptors in bosentan-treated SCD mice.24 Thus, our data highlight the importanceof selectively targeting the ETA receptor to achieve maximalrenal protection in SCD.

Recent studies have re-emphasized the role of proximaltubular uptake of albumin in protection against albumin-uria.49,50 Renal tubules are known to be highly susceptible tohypoxic conditions51 and it has been reported that urinaryKIM-1 and NAG levels, biomarkers of tubular injury, stronglycorrelated with albuminuria in patients with SCD.52 There-fore, we investigated the contribution of proximal tubularinjury to albuminuria in SCD. We found that selective ETAreceptor antagonism protects against tubular injury in HbSSmice, which may contribute to the antiproteinuric effect ofthe ETA receptor antagonist. These observations are also inagreement with those reported by Hocher et al.53 and maysuggest an additional benefit of the use of ETA receptor an-tagonists in the treatment of sickle nephropathy. Our resultssuggest that renal tubular protein uptake may involve ETB

receptor activation. Although proteinuria is often presumedto be a result of glomerular damage in SCD,54 the question ofwhether tubular injury contributes to impaired albuminuriaor whether tubular injury is the consequence of a continualprotein load remains unanswered. One explanation of thelack of reduction in albuminuria and proteinuria after ad-ministration of the dual ETA/B receptor antagonist in HbSS

magnification,340 (scale bar=50 mm). (B) Depicts the quantification of (A) represented as the average number of WT-1–positive cells perglomerulus. Podocytes were counted in minimum 20 glomeruli in ten sections per slide. Data are mean6SEM; n=5 in HbAA and HbSSgroups. (C) Depicts the relative WT-1 mRNA expression in glomeruli after 10-week treatment protocol. (D) Depicts the relative nephrinmRNA expression in glomeruli after 10-week treatment protocol. (E) Depicts the relative podocin mRNA expression in glomeruli after10-week treatment protocol. (F) Depicts the relative synaptopodin mRNA expression in glomeruli after 10-week treatment protocol. (G)Depicts representative photomicrographs of transmission electron microscopy sections of glomeruli. Data are mean6SEM; *P,0.05versus vehicle-treated HbAA; #P,0.05 versus vehicle-treated HbSS; n=5–6 in HbSS and n=6 in vehicle-treated HbAA group.



mice may involve increased albumin endocytosis in theproximal tubule followed by cell proliferation and injury.A second hypothesis is that blocking of ETB receptors in

renal proximal tubules may cause a decrease in tubular al-bumin uptake, an increase in urinary albumin excretion, andultimately tubular apoptosis and injury. This hypothesis is

Figure 4. Evidence from isolated glomeruli suggests that ETA receptor antagonism prevents the onset of glomerular inflammation and oxidativestress in humanized sickle cell mice. Expression of components of the ET system, inflammation, and oxidative stress in glomeruli from geneticcontrol (HbAA) and humanized sickle mice (HbSS) treated with vehicle, the selective ETA antagonist, ambrisentan, or the combined ETA/B an-tagonist, A-182086, for 10 weeks beginning at 4 weeks of age. (A) Depicts the relative ET-1 mRNA expression in glomeruli after 10-weektreatment protocol. (B) Depicts the relative ETA receptor mRNA expression in glomeruli after 10-week treatment protocol. (C) Depicts the relativeETB receptor mRNA expression in glomeruli after 10-week treatment protocol. (D) Depicts the relative P-selectin mRNA expression in glomeruliafter 10-week treatment protocol. (E) Depicts the relative vascular cell adhesion molecule 1 (VCAM-1) mRNA expression in glomeruli after 10-week treatment protocol. (F) Depicts the relative monocyte chemoattractant protein-1 (MCP-1) mRNA expression in glomeruli after 10-weektreatment protocol. (G) Depicts the glomerular ROS production after 10-week treatment protocol. Data are mean6SEM; *P,0.05 versus vehicle-treated HbAA; #P,0.05 versus vehicle-treated HbSS; n=5–6 in HbSS and n=6 in vehicle-treated HbAA group. AUC, area under the curve.



supported by several studies implicatingET-1 as proapoptotic in the kidney.55–58

Our results demonstrate that, althoughthe nonselective ETA/B receptor antago-nist improved glomerular structure andfunction to some extent in HbSS mice,increased urinary albumin excretion wasstill present, thus excluding glomerularinjury as the only cause of albuminuria.Also, the observed downregulation of mega-lin and FcRn may be related to the oxidativestress–mediated transcription factor, NFkB,activation and facilitated release of proin-flammatory and profibrotic mediators, in-cluding MCP-1 or P-selectin.59 These resultsare also consistent with the protective role ofETB receptor in proximal tubules. Moreover,data from our laboratory has shown aprotective role of ETB receptor activationin tunicamycin-induced renal tubularapoptosis.60 Thus, our results suggestthat the albuminuria observed in HbSSmice treated with the dual ETA/B receptorantagonist is a combination of glomeru-lar and tubular injury or dysfunction.

In conclusion, this study implicates theETA receptor as an important mediator inthe development and progression of renal

Figure 5. Long-term ETA receptor antagonism prevents renal tubular injury in humanizedsickle cell mice. Histologic examination of the renal cortex and urinary excretion of markersof tubular injury of genetic control (HbAA) and humanized sickle mice (HbSS) treated withvehicle, the selective ETA antagonist, ambrisentan, or the combined ETA/B antagonist, A-182086, for 10 weeks beginning at 4 weeks of age. (A) Depicts the average urinary KIM-1

excretionafter10-weektreatmentprotocol. (B)Depicts the average urinary NAG excretionafter 10-week treatment protocol. (C) DepictstheaverageurinaryNGALexcretion, iron-bindingprotein, after 10-week treatment protocol. Dataaremean6SEM; *P,0.05 versus vehicle-treatedHbAA; #P,0.05 versus vehicle-treated HbSS;n=9–10 in HbSS and n=9 in untreated HbAAgroup. (D) Depicts brush border thickness indexscore. (E) Depicts interstitial fibrosis index score.(F)Depictsquantificationof irondeposition in thewhole kidney sections (megapixel per microme-ter). (G) Depicts representative periodic acidSchiff–hematoxylin–stained sections of renalcortex. Original magnification, 340 (scalebar=50 mm). (H) Representative Massontrichrome–stained sections of renal cortex andmedulla. Original magnification, 310 (scalebar=200 mm, respectively). (I) Depicts Prussianblue iron–stained sections of renal cortex. Origi-nal magnification, 320 (scale bar=100 mm). Fi-brosis and brush border thickness were assessedin ten sections per slide. Data are mean6SEM;*P,0.05 versus vehicle-treated HbAA; #P,0.05versus vehicle-treated HbSS; n=5 in HbAA andHbSS groups.



Figure 6. Selective ETA receptor antagonism prevents renal cortical inflammation and immune cell infiltration in humanized sickle cellmice. Immunohistochemical examination and mRNA expression of proinflammatory and profibrotic markers in renal cortex of geneticcontrol (HbAA) and humanized sickle mice (HbSS) treated with vehicle, the selective ETA antagonist, ambrisentan, or the combinedETA/B antagonist, A-182086, for 10 weeks beginning at 4 weeks of age. (A) Depicts representative CD3+–stained cortical sections.Original magnification, 340 (scale bar=50 mm). (B) Depicts the quantification of glomerular CD3+ cells represented as the average



injury in SCD, and suggests that selective ETA receptor antag-onism is sufficient to prevent development and progression ofrenal injury in SCD. These findings provide rigorous proof-of-concept evidence to support the use of ETA receptor antago-nism in the treatment of sickle nephropathy, and by utilizing awell tolerated and clinically approved drug in our treatmentstrategy, the barriers to the next steps of clinical investigationare minimal.

CONCISE METHODS

AnimalsStudies used humanized SCD knock-in mice, with notation:

B6;129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB*)Tow/Hbbtm3(HBG1,HBB)Tow/J,

developed by Townes et al.61 Experimental animals (HbSS) were ho-

mozygous for mutant b globin (E to V at position 6) and expressed

humanHbS. Control animals (HbAA), derived from the same colony,

were homozygous for wild-type b globin and expressed humanHbA.

All studies were performed in 14-week-old HbSS mice with similar

numbers of males and females in each group and age-matched ge-

netic controls. Group numbers varied depending on availability of

mice from the breeding colony. All mice were housed under condi-

tions of constant temperature, humidity, and 12-hour light/dark

cycle, and were provided with food (Harlan Teklad) and water ad

libitum. Separate groups of mice were placed in standard metabolic

cages for 48 hours before the experiments. Mice were allowed to

adapt to metabolic cages for 1 day before collection of 24-hour urine

samples. All mice were maintained and studied in accordance with

the National Institutes of Health Guide for the Care and Use of

Laboratory Animals following a protocol reviewed and approved

by the University of Alabama at Birmingham Institutional Animal

Care and Use Committees.

Drug Treatment ProtocolsThe selective ETA receptor antagonist ambrisentan (10mg/kg per day;

AbbVie, Inc., Abbott Park, IL), nonselective ETA/ETB receptor antag-

onist A-182086 (10mg/kg per day; AbbVie, Inc., Abbott Park, IL), or

vehicle were administrated in drinking water and the concentrations

were adjusted daily according to the water intake, as previously de-

scribed.62 To assess the treatment of established nephropathy, antag-

onists were administrated for 2 weeks beginning at 12 weeks of age,

and this strategy was referred to as the 2-week treatment protocol. To

assess ability of treatment to prevent the onset of nephropathy,

antagonists were administrated for 10 weeks beginning at 4 weeks

of age (time of weaning), and this strategy was referred to as the

10-week treatment protocol.

BP MeasurementsStandard tail-cuff BP measurements, using a pneumatic transducer,

were obtained on five consecutive days from 13-week-old HbSS and

HbAAmice that had undergone 5 days of prior training as previously

described.63 Data are presented as the average of five measurements

for each animal.

GFR MeasurementsGFR was measured in 14-week-old HbSS and HbAA mice (1 day

before metabolic cage studies) using transcutaneous measurement

of fluorescein isothiocyanate–labeled sinestrin technique as previ-

ously described.64

Measurements of PalbGlomeruli were isolated on ice by consecutive sieving technique as

previously described with minor modifications.65,66 Briefly, renal

cortical tissue was passed through a series of filters (with pore size

180, 100, and 50 mm) resulting in suspension of decapsulated, intact

glomeruli in ice-cold Hanks Balanced Salt Solution, pH 7.4 (Corning,

NY). Freshly isolated glomeruli were used formeasurements of Palb as

previously described.66–68 In brief, change in glomerular volume was

measured in response to an oncotic gradient induced by defined con-

centrations of albumin in the buffer solution.

Glomerular ROS AssayGlomerular ROS production was measured using a luminescence

assay as previously described.22,28

Plasma and Urine AnalysesAll urine analyses were performed on 24-hour urine samples collected

inmetabolic cages.Urinary protein concentrationwasmeasuredusing

the Bradford assay (BioRad Laboratories, CA). Urinary albumin con-

centrationwas determined using an immunoperoxidase assay accord-

ing to the manufacturer’s instructions (GenWay Biotech Inc., CA).

Urinary nephrin concentration was measured using a mouse NPHN

ELISA kit (Uscn Life Science Inc., TX). Urine levels of tubular injury

markers were measured using ELISA for mouse KIM-1 (ab119596-

KIM-1 [TIM-1] Mouse ELISA kit; Abcam, UK), NAG (Mouse NAG

kit; Crystal Chem, IL), and NGAL (Lipocalin-2 [NGAL] Mouse

ELISA kit; Abcam, UK). Plasma levels of ET-1 were determined by

number of CD3+ cells per glomerulus (minimum20 glomeruli counted). (C) Depicts the quantification of cortical nonglomerular CD3+ cellsrepresented as the averagenumber of nonglomerularCD3+ cells per field. (D)Depicts representativeF4/80+-stained cortical sections.Originalmagnification,340 (scale bar=50 mm). Arrows indicate F4/80+ cells. (E) Depicts the quantification of glomerular F4/80+ cells represented asthe average number of F4/80+ cells per glomerulus (minimum 20 glomeruli counted). (F) Depicts the quantification of cortical nonglomerularF4/80+ cells represented as the average number of nonglomerular F4/80+ cells per field. CD3+ and F4/80+ cells were assessed in ten sectionsper slide andcalculated.Data aremean6SEM;n=5 inHbAAandHbSSgroups. (G)Depicts the relativeMCP-1mRNAexpression in cortex after10-week treatment protocol. (H) Depicts the relative P-selectin mRNA expression in cortex after 10-week treatment protocol. (I) Depicts therelative VCAM-1 mRNA expression in cortex after 10-week treatment protocol. Data are mean6SEM *P,0.05 versus vehicle-treated HbAA;#P,0.05 versus vehicle-treated HbSS; n=5–6 in HbSS and n=6 in vehicle-treated HbAA group.



Figure 7. Analysis of tubular albumin transporters reveals that ETA receptor antagonism preserves megalin expression in the renal cortex ofhumanized sickle cell mice. Expression of receptors that participate in albumin handling from renal cortex of genetic control (HbAA) andhumanized sickle mice (HbSS) treated with vehicle, the selective ETA antagonist, ambrisentan, or the combined ETA/B antagonist, A-182086, for10 weeks beginning at 4 weeks of age. (A) Depicts the relative megalin mRNA expression in cortex after 10-week treatment protocol. (B)Depicts Western blot analysis of megalin expression in cortical extracts. (C) Depicts quantification of Western blot bands for megalin. (D)Depicts the relative cubilin mRNA expression in cortex after 10-week treatment protocol. (E) Depicts the relative amnionless (AMN) receptormRNA expression in cortex after 10-week treatment protocol. (F) Depicts the relative FcRn mRNA expression in cortex after 10-week treatmentprotocol. Data are mean6SEM; *P,0.05 versus vehicle-treated HbAA; #P,0.05 versus vehicle-treated HbSS; n=5–6 in HbSS and n=6 invehicle-treated HbAA group. a-18, A-182086; Amb., Ambrisentan; MW, molecular weight; RDU, relative densitometry units; Veh., vehicle.



ELISA according to manufacturer’s instructions (QuantiGlo ET-1

Kit; R&D Systems, MN). University of Alabama at Birmingham–

University of California San Diego O’Brien Center for Acute Kidney

Injury Research bioanalytic core facility measured plasma creatinine

levels by liquid chromatography–tandem mass spectrometry.

Histologic AnalysisKidneys isolated from 10-week treatment protocol mice were im-

mersed in 10% formalin and embedded in paraffin. Kidney sections

(4-mm thick) were processed for histopathology studies and im-

munohistochemistry assay. Sections were stained with hematoxylin

and eosin, Masson trichrome, periodic acid Schiff–hematoxylin,

or Prussian blue using standard protocols. Tissues were evalu-

ated blindly according to the criteria used for quantification of

the changes in renal structures, as previously described.69–72

Briefly, the extent of glomerular sclerosis was assessed on Masson

trichrome–stained sections using a grading score from 0 to 4: 0 for

no sclerosis; 1 for sclerosis in up to 25% of the glomerulus; 2 for

sclerosis in up to 50% of the glomerulus; 3 for sclerosis in up to 75%

of glomerulus; and 4 for sclerosis in up to 100% of the glomerulus.

Glomerular basement membrane thickening score was assessed us-

ing the following 0–4 scale: 0 for no basement membrane thicken-

ing; 1 for mild thickening; 2 for moderate thickening; 3 for severe

thickening; and 4 for massive thickening. Glomerular vascular con-

gestion was assessed on hematoxylin and eosin–stained sections and

calculated as the percentage of total glomeruli with congestion

present in at least 25% of the glomerulus. A minimum of 20 glo-

meruli were evaluated under 4003 magnification from ten differ-

ent bright-field regions of renal cortex and results were averaged for

each kidney. Tubular brush border thickness was assessed on peri-

odic acid Schiff–hematoxylin–stained sections using a 0–4 grading

scale: 0 for no changes; 1 for lesions involving ,25% of the area;

2 for lesions involving 25%–50% of the area; 3 for lesions involving

.50% of the area; and 4 for lesions involving nearly 100% of the

area. Results were averaged for each animal. Tubulointerstitial fi-

brosis was assessed in ten fields per kidney section and graded as

follows: 0 for no fibrosis; 1 for mild fibrosis; 2 for moderate fibrosis;

3 for severe fibrosis; and 4 for fibrosis involving nearly the entire

area, and results averaged for each animal. Renal iron deposition

was assessed on scanned images of whole kidney by MetaMorph

software (MetaMorph; Molecular Devices LLC, CA).

For immunohistochemistry, sections were stained forWT-1 using

anti–Wilms tumor protein antibody (CAN-R9[IHC]-56–2; ab89901,

Abcam, UK), anti-CD3 (T cells) antibody (ab16669,1:600; Abcam,

UK), and anti-F/4/80 (macrophages) antibody (MCA497GA, 1:200;

BioRad). WT-1–positive cells were counted under 4003 magnifica-

tion from a minimum of 20 glomeruli from ten different bright-field

regions of the cortex and results were averaged for each kidney. Renal

cortical CD3+ and F4/80+ cells were blindly quantified in ten micro-

scopic fields (200 3 200 mm, 4003 magnification) and results were

averaged for each kidney.

Electron MicroscopySmall cubes of renal cortex (1.53 1.53 1.5 mm) were drop-fixed in

2.5% glutaraldehyde/2.5% formalin, postfixed in 1% osmium

tetroxide, and processed into resin blocks. Ultrathin sections were

cut using a diamond knife, placed in a copper grid, stained with

uranyl acetate and lead citrate, and examined using an FEI Tecnai

T12 Spirit 20–120 kv transmission electron microscope.

Total RNA Extraction and Quantitative Real Time PCRTotal RNA extraction of renal cortex or glomeruli was performed

using Ambion PureLink RNA Mini Kit or RNAqueous-Micro Total

RNA Isolation Kit (Ambion, Austin, TX), and reverse transcription

with the iScript cDNA synthesis kit (BioRad, Hercules, CA). Real-

time amplification was performed with ABI 7300 Real-Time PCR

System using iTaq Universal Probes Supermix (BioRad, Hercules,

CA) and TaqMan primer gene expression assay (Applied Biosystems,

Foster City, CA) of ET-1 (Mn00438656_m1), ETA (Mn01243722_m1),

ETB (Mn00432989_m1),WT-1 (Mn01337048), nephrin (Mn00497828),

podocin (Mn01292252_m1), synaptopodin (Mn03413333_m1), MCP-1

(Mn00441242_m1), P-selectin (Mn01295931_m1), V-CAM1

(Mn01320970_m1), megalin (Mn01328171_m1), cubilin

(Mn01325077_m1), amnionless (AMN; Mn00473870_m1), and

FcRn (Mn01205451_m1) according to the manufacturer’s instruc-

tions. The comparative method of relative quantification (2-DDCT)

was used to calculate the expression level of each target gene, nor-

malized to b-actin. Data are presented as the relative mRNA expres-

sion of the specific gene of interest.

Western BlottingThirty micrograms of protein was electrophoresed on 4%–15% gels

(#4561086; Bio-Rad) as previously described.73 Blots were probed

with a rabbit polyclonal antibody against the megalin c-terminus

(ab76969; Abcam, UK). Equivalent protein loading was confirmed

by Coomassie staining.

Statistical AnalysesAnalyseswereperformedusingGraphPadPrism6.0 software (GraphPad

Software Inc., CA). Data were compared using paired t test or two-way

ANOVAwith Bonferroni post hoc correction (variables: genotype, treat-

ment). One-way ANOVA with Bonferroni’s post hoc correction for

multiple comparisons was utilized for Western blot data. Results are

expressed as mean6SEM with a=0.05.

ACKNOWLEDGMENTS

The technical assistance of Binli Tao and Kaquanta Barlow is greatly

appreciated. The authors also thank Xiaofen Liu and Ping Hua for

assistance with histologic and Western blot studies. We acknowledge

the University of Alabama at Birmingham High Resolution Shared

Imaging Facility for assistancewith electronmicroscopy, as well as the

Center for Metabolic Bone Disease Core Laboratory for assistance in

preparing tissues for histologic evaluation.

This research was supported by grants from the National Heart,

Lung, and Blood Institute, U01 HL117684 to A.K., J.S.P., and D.M.P.;

K99 1K99HL127178 and T32DK079339 to J.S.S.; National Institutes

ofHealth T32DK007545 toC.D.M.; theNational Institute ofDiabetes

and Digestive and Kidney Disease, F30 DK107194 to B.M.F.; the



American Heart Association, 15POST25090329 to E.Y.G.; the

American Society of Nephrology, Joseph A. Carlucci Research

Fellowship to M.K. and P30 DK079337 (the core resource of the

University of Alabama at Birmingham–University of California San

Diego O’Brien Center).

Parts of this work were presented during Kidney week, November

7, 2015, San Diego, CA, and Kidney Week, November 18, 2016,

Chicago, IL.

DISCLOSURESNone.

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See related editorial, “Endothelin-A Receptor Antagonism Retards the Pro-gression of Murine Sickle Cell Nephropathy,” on pages 2253–2255.

This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2016070711/-/DCSupplemental.





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