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Therapeutic Inhibition of VEGF Signaling and Associated Nephrotoxicities

Chelsea C. Estrada,1 Alejandro Maldonado,1 and Sandeep K. Mallipattu1,2

1Division of Nephrology, Department of Medicine, Stony Brook University, Stony Brook, New York; and 2Renal Section, Northport Veterans Affairs Medical Center, Northport, New York

ABSTRACT Inhibition of vascular endothelial A (VEGFA)/vascular endothelial with and proteinuria. Re- growth factor 2 (VEGFR2) signaling is a common therapeutic strategy in ports describe histologic changes in the oncology, with new continuously in development. In this review, we consider kidney primarily as glomerular endothe- the experimental and clinical evidence behind the diverse nephrotoxicities associ- lial injury with thrombotic microangiop- ated with the inhibition of this pathway. We also review the renal effects of VEGF athy (TMA).8 has inhibition’s mediation of key downstream signaling pathways, specifically MAPK/ also been observed,9 with the clinical ERK1/2, endothelial nitric oxide synthase, and mammalian target of rapamycin manifestations varying according to (mTOR). Direct VEGFA inhibition via binding or VEGF trap (a soluble decoy mechanism and direct target of VEGF receptor) is associated with renal-specific thrombotic microangiopathy (TMA). Re- inhibition. ports also indicate that inhibition of the VEGF receptors is prefer- Current VEGF inhibitors can be clas- entially associated with glomerulopathies such as minimal change disease and FSGS. sifiedbytheirtargetofactioninthe Inhibition of the downstream pathway RAF/MAPK/ERK has largely been associated VEGFA-VEGFR2 pathway: drugs that with tubulointerstitial injury. Inhibition of mTOR is most commonly associated with bind to VEGFA, sequester VEGFA, in- albuminuria and podocyte injury, but has also been linked to renal-specificTMA.In hibit receptor tyrosine (RTKs), all, we review the experimentally validated mechanisms by which VEGFA-VEGFR2 or inhibit downstream pathways. A crit- inhibitors contribute to nephrotoxicity, as well as the wide range of clinical manifes- ical review of VEGFA-VEGFR2 signaling tations that have been reported with their use. We also highlight potential avenues in the kidney is necessary to fully under- for future research to elucidate mechanisms for minimizing nephrotoxicity while standthe mechanismsresponsible for the maintaining therapeutic efficacy. nephrotoxicity associated with the onco- logical use of VEGF inhibition. J Am Soc Nephrol 30: ccc–ccc, 2019. doi: https://doi.org/10.1681/ASN.2018080853

VEGF SIGNALING IN THE KIDNEY

Inhibitors of vascular development were of , a recombinant IgG Filtration of plasma in nephrons occurs fi rst investigated as potential chemother- mAb against VEGFA, inhibiting VEGF in the glomerular capillary beds at the fi apeutic agents on the basis of study nd- signaling proved to be a promising ap- glomerular filtration barrier, which con- ings from the late 20th century regarding proach to halting neoplastic develop- sists of three layers: fenestrated endothe- 5 the critical role of in tumor ment. Since then, many additional lial cells, , and the development and growth.1 Subsequent agents that block VEGF signaling and investigations led to the discovery of its downstream pathways have become vascular endothelial growth factor A available for the treatment of various (VEGFA), an essential growth factor (Figure 1, i–vi). Published online ahead of print. Publication date for angiogenesis.2,3 Previous studies by Although these agents offer substan- available at www.jasn.org. Kim et al.4 used murine cell lines tial benefit, both in the treatment of Correspondence: Dr. Sandeep K. Mallipattu, Department to show that treatment with an mAb many solid tumors6 as well as age- of Medicine/Nephrology, Stony Brook University, 100 Nicolls Road, HSCT16-80E, Stony Brook, NY 11794- 7 against VEGFA decreased tumor growth, related , their use 8176. Email: sandeep.mallipattu@stonybrookmedicine. indicating a potential therapeutic role is associated with significant nephrotoxi- edu 8 for VEGFA inhibition in cancer treat- city. Most commonly, pharmacologic Copyright © 2019 by the American Society of ment. Beginning with the development VEGF inhibition has been associated Nephrology

J Am Soc Nephrol 30: ccc–ccc, 2019 ISSN : 1046-6673/3002-ccc 1 REVIEW www.jasn.org

P P

i. VEGFA Ab VEGFA (Bevacizumab, ii. VEGF Trap ) ()

GEnC iii. VEGFR2 Ab () GBM FP VEGFR2 iv. Inhibitors (, FP , , ) RAS/MAPK/ ERK Pathways

v. BRAF Inhibitors GEnC BRAF EF (, Pl3 Kinase/ ) AKT Pathways

eNOS MTORC1 vi. mTOR Inhibitors EF (, , )

Figure 1. VEGFA-VEGFR2 signaling pathways and their pharmacological inhibition occur across the glomerular filtration barrier. VEGFA is released from podocytes and binds to its receptor (VEGFR2) on glomerular endothelial cells. (i) Bevacizumab and ranibizumab are mAbs against VEGFA and inhibit angiogenesis through IgG antibody interaction with all of its isoforms. (ii) Aflibercept is a recombinant comprising binding domains for VEGFR1 and VEGFR2 attached to the Fc portion of human IgG1, and acts as a soluble decoy receptor or “VEGF trap.” (iii) Ramucirumab is a fully humanized IgG1 mAb that specifically inhibits VEGFR2 by targeting its extracellular domain. (iv) TKIs such as sunitinib, , , and axitinib target VEGFR2, as well as interfere with the activity of additional RTKs such as PDGF receptor, fibroblast , and EGF receptor, which all share a similar structure. (v) Agents such as vemurafenib and dabrafenib have been recently developed to specifically target B-Raf, a component of the intracellular MAPK/ERK intracellular pathway. (vi) mTOR inhibitors such as temsirolimus, ridaforolimus, and everolimus are used across several malignancies and act downstream of the phosphatidylinositide 3-kinase (PI3K)/AKT signal transduction pathway. Ab, antibody; BRAF, B-Raf Proto-Oncogene; EF, endothelial fenestrations; FP, foot process; GBM, glomerular basement membrane; GEnC, glomerular endothelial cell; P, Podocyte. foot processes of visceral epithelial cells form, which acts as a decoy receptor, in- wide range of glomerulopathies (Table 1) or podocytes. In the kidney, VEGFA is hibiting VEGFA signaling,17 and is the sole demonstrates that tight regulation of expressed by both podocytes10 and renal receptor for VEGFB.12 VEGFA signaling in the kidney is critical tubular epithelial cells11;inhumans, The functional diversity of VEGF re- to glomerular development and the main- VEGFA165 is the most abundant iso- ceptors was initially elucidated by the tenance of mature glomerular function in form.12 VEGFA binds to one of two creation of knockout mice. Mice defi- both homeostasis and disease. For example, RTKs, VEGFR1 and VEGFR213; although cient in Veg fr2 die in utero from a defect knockout of Veg fa during embryogenesis— both receptors have been identified as in hematopoietic and endothelial cell de- including global homozygous or heterozy- playing an important role in angiogenesis, velopment18; embryonic lethality of gous knockout22,23 or podocyte-specific VEGFR2 has been more extensively stud- Veg fr1 deletion is caused by endothelial knockout10—is uniformly lethal at or be- ied, as it is responsible for a majority of cell overgrowth and disorganization.19 fore birth. Mice with podocyte-specificpar- VEGFA signaling and is abundantly dis- These whole-body knockout mice un- tial deletion of Veg fa survive the perinatal tributed in stromal and malignant vascular derscore the key role of VEGF signaling period, but develop endotheliosis and renal tissues.14,15 Both receptors are primarily in endothelial cell proliferation, migra- failure by 9 weeks of age.10 localized in the glomerular and peritubu- tion, and permeability.20 In adult mice, inducible podocyte-specific lar capillary .11,16 Further- The association of VEGFA overex- Veg fa deletion produces renal-specific more, VEGFR1 also exists in a soluble pression10 or reduction8,21,10 with a TMA, which recapitulates kidney biopsy

2 Journal of the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc,2019 www.jasn.org REVIEW

Table 1. Renal manifestations in VEGF-VEGFR transgenic murine models Genotype Model Effects Reference 2 2 Vegfa / Constitutive, whole-body Embryonically lethal 22,23 deletion of Vegfa 1 2 Vegfa / Constitutive, whole-body partial Embryonically lethal between days 11 22,23 deletion of Vegfa and 12; defective yolk sac blood supply 2 2 Vegfr1 / Constitutive, whole-body Embryonically lethal because of 19 deletion of Vegfr1 endothelial cell overgrowth and disorganization 2 2 Vegfr2 / Constitutive, whole-body Embryonically lethal because of defect in 18 deletion of Vegfr2 hematopoietic and endothelial cell development Nephrin-Cre; Vegfaflox/flox Constitutive deletion of Vegfa Perinatally lethal, mice die at birth or 10 from podocytes within 18 h with small glomeruli with few capillary loops 1 Nephrin-Cre; Vegfaflox/ Constitutive, partial deletion of Vegfa Endotheliosis, with eventual 10 from podocytes glomerulosclerosis and ESRD by 9–12 wk of age Podocin-rtTA; Doxycycline-inducible deletion of Vegfa Renal thrombotic microangiopathy 8 TetO-Cre; Vegfaflox/flox in podocytes Pax8-rtTA; Doxycycline-inducible deletion of Vegfa Small, histologically normal kidneys with 11 TetO-Cre; Vegfaflox/flox in tubular cells peritubular capillary rarefaction Rosa-rtTA; Doxycycline-inducible whole-body Glomerular endothelial injury and 24 TetO-Cre; Vegfr2flox/flox deletion of Vegfr2 ascites by 2.5 mo of age Nephrin-Cre; Vegfr2flox/flox Constitutive deletion of Vegfr2 Normal glomeruli and intact 24 from podocytes glomerular filtration barrier 10 Nephrin-VEGFA164 Constitutive overexpression of the Collapsing glomerulopathy within 5 d of age 164 isoform of VEGFA in podocytes Podocin-rtTA: Doxycycline-inducible overexpression of Glomerulomegaly, mesangial expansion, 28

TetO-VEGF164 the 164 isoform of VEGFA in podocytes foot process effacement

findings in individuals treated with podocyte autocrine dependence on se- glomerular endothelial cells to alterations VEGF inhibitors.8 In contrast, mice creted VEGFA, were corroborated in cul- in VEGFA-VEGFR2 signaling.27 with tubule-specific deletion of Veg fa tured human podocytes treated with the Increased VEGFA-VEGFR2 signaling had histologically normal kidneys with BRAF inhibitor dabrafenib and the alsoappearstohavedetrimentalglomerular some peritubular capillary density MEK1 inhibitor , which effects. Constitutive overexpression of 11 loss, emphasizing the essential role of strongly inhibited VEGF release and simul- Veg fa164 in podocytes leads to collapsing podocyte-derived Veg fa.Instudiesof taneously increased albumin permeabil- glomerulopathy,10 whereas its inducible knockout mice, Sison et al.24 highlighted ity.26 Furthermore, biopsy specimens of overexpression results in glomerulome- the importance of paracrine VEGFA- patients treated with this combination galy with mesangial expansion.28 Taken VEGFR2 signaling between the podocyte exhibited severe podocyte injury together, these findings suggest that and endothelial cell, showing that mice and effacement.26 maintenance of glomerular endothelial with podocyte-specific deletion of Vegfr2 Interestingly, in addition to developing integrity relies heavily on tight regulation did not develop glomerulopathy, but renal-specific TMA, mice with inducible of paracrine VEGFA-VEGFR2 signaling those with whole-body inducible dele- podocyte-specific Veg fa deletion exhibited between the podocyte and renal endothe- tion of Veg fr2 developed TMA, resem- reduced glomerular complement factor H lium, and that administration of antian- bling mice lacking podocyte-specific (CFH) staining and increased glomerular giogenic therapeutics to disrupt this Veg fa. C3 deposition.27 The dependence of the signaling and its downstream pathways Although these experiments deempha- expression of the complement regulatory directly results in renal endothelial in- sized autocrine VEGFA-VEGFR2 signal- protein CFH on VEGFAwas also shown in jury, manifested primarily as protein- ing in the podocyte, other studies have cultured glomerular endothelial cells, uria, hypertension, and renal-specific suggested the contrary. In cultured mouse where exogenous VEGF directly increased TMA.8,29,30 However, it should be noted podocytes, VEGFA treatment reduced ap- CFH expression.27 This relationship was that histologic diagnosis of such injury is optosisaswellasupregulatedpodocinpro- not seen in other endothelial cell lines, often limited because of the underutili- tein expression.25 These results, suggesting perhaps explaining the sensitivity of zation of kidney biopsies. In addition,

J Am Soc Nephrol 30: ccc–ccc, 2019 VEGF Inhibition and Nephrotoxicity 3 REVIEW www.jasn.org these manifestations occur with variable showed lesions of TMA by histology, likely an early hypertensive response on clinical onset after therapy initiation, are not reflecting the short duration of treatment; outcomes after bevacizumab treatment.42 dose-related, and are often reversible. however, the significant glomerular injury However, in patients receiving ramuciru- seen in both models underscores the im- mab for gastric cancer, treatment-related portance of renal VEGF signaling during hypertension was predictive of improved THERAPEUTIC VEGF INHIBITION development as well as in homeostasis. outcomes.43,44 Findings from animal models demon- Importantly, Eremina et al. reported Classes of VEGF inhibitors act through strate the importance of VEGF signaling that in a case series of six patients, bevaci- different mechanisms to cause endo- during renal injury. In a rat model of cres- zumab use was associated with develop- thelial and glomerular injury. In the centic GN, direct VEGF inhibition via an ment of renal TMA. The patients had following section, we review the experi- intramuscular injection of the soluble re- hypertension, proteinuria, and biopsy mentally validated mechanisms by ceptor for VEGFR1 (sFlt-1 plasmid), ad- findings of glomerular endothelial cell in- which VEGFA-VEGFR2 inhibitors con- ministered 3 days before injection of jury (Table 3)—findings the investigators tribute to nephrotoxicity. nephrotoxic serum, exacerbated crescent confirmed in a transgenic mouse model formation and albuminuria.35 Similarly, (Table 1).8 This association was also noted subcutaneous injection of dRK6, which in a large biopsy series, in which 66 out of ANTI-VEGF MAB binds to VEGFA, worsened albuminuria 67 (98.5%) patients who received either and podocyte injury in db/db diabetic bevacizumab or aflibercept had renal Pharmacologic agents that inhibit VEGFA mice.36 Both studies demonstrated the TMA; the remaining patient had FSGS.9 activity through antibody binding include importance of VEGF signaling in main- Of those patients with biopsy-proven bevacizumab, ranibizumab, aflibercept, and taining glomerular integrity in disease as TMA, mean onset of disease was 6.9 ramucirumab. Bevacizumab and ranibizu- well as in homeostasis. months from the start of treatment; mab are mAbs against VEGFA and inhibit Thenephrotoxicitiesassociatedwithdi- 83.5% had hypertension, and mean daily angiogenesis through IgG antibody interac- rect VEGF inhibition in patients receiving protein excretion was subnephrotic, at tion with all of its isoforms31 (Figure 1i). chemotherapeutics is primarily derived 2.5 g/d.9 In contrast to the effects of sys- Aflibercept, a recombinant fusion protein from data (Table 3). In a temic anti-VEGF therapy, intraocular comprising binding domains for VEGFR1 meta-analysisof20phase2andphase3 anti-VEGF mAbs for the treatment of di- and VEGFR2 attached to the Fc portion clinical trials involving bevacizumab- abetic typically has not been of human IgG1, acts as a soluble decoy based therapy in solid tumors, the inci- associated with these adverse effects.45 receptor, or VEGF trap (Figure 1ii).32 dence of all-grade hypertension was However, there have been case reports of Ramucirumab, a fully humanized IgG1 23.6% and high-grade (grade 3 or 4) hy- TMA with microangiopathic hemolytic mAb, specificallyinhibitsVEGFR2bytar- pertension was 7.9%.37 Similarly, a meta- anemia and after in- geting its extracellular domain (Figure 1iii). analysis of seven randomized, controlled travitreal ranibizumab,46 and of onset of Initial models of the first-in-class agent trials (RCTs) found the incidence of pro- proteinuria and antibody-mediated rejec- bevacizumab measured efficacy by reduced teinuria subsequent to bevacizumab ther- tion in kidney transplant patients after in- tumor growth using human apy ranged from 21% to 62%, with the travitreal bevacizumab, ranibizumab, and cell lines implanted into nude mice,33 and greatest risk associated with higher-dose aflibercept.47 In all, across multiple animal the adverse outcomes of proteinuria and therapy.38 Data from six phase 2 trials models, as well as in clinical trials, direct hypertension were not observed until phase showed that the incidence of all-grade hy- and systemic VEGF inhibition, via anti- 1trials.34 In murine models of direct VEGF pertension and proteinuria after ramucir- body binding or VEGF trap, results in hy- inhibition in wild-type mice (Table 2), a umab therapy for solid tumors was 21% pertension, proteinuria, and a spectrum single intravenous dose of anti-VEGF anti- and 9%, respectively.39 A meta-analysis of of glomerular endothelial injury. body produced significant albuminuria 15 trials of aflibercept use in colorectal after 3 hours, as well as glomerular endo- cancer found the incidence of all-grade Tyrosine Kinase Inhibitors of thelial cell hypertrophy and disruption of and high-grade hypertension to be VEGF Signaling glomerular basement membrane, seen by 42.4% and 17.4%, respectively; the risk Tyrosine kinase inhibitors (TKIs) inhibit electron microscopy.17 In validation of ge- of developing hypertension was signifi- RTKs, which consist of an extracellular netic knockout studies, mice given antibody cantly higher with aflibercept treatment bindingdomain,atransmembraneregion, 40 directed at recombinant VEGF165 during compared with bevacizumab treatment. and an intracellular kinase that mediates the neonatal period (on days 0, 2, 4, and Although some researchers have suggested signal transduction. TKIs interfere with 5) displayed impaired glomerulogenesis, that hypertension subsequent to VEGF in- the activity of one or more families of with poor cellularity and increased extracel- hibition is correlated with improved re- RTKs, including VEGFR, PDGF receptor lular matrix deposition,21 thereby confirm- sponse to therapy,41 a meta-analysis of (PDGFR), fibroblast growth factor recep- ing the essential role of VEGF signaling in seven phase 3 RCTs across multiple tumor tor, and EGF receptor, which all share a glomerular development. Neither model types found no predictive significance of similarstructure. TKIs are thus commonly

4 Journal of the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc,2019 mScNephrol Soc Am J Table 2. Renal manifestations from pharmacologic VEGF inhibition in murine models Animal Model (Model/ Dose, Route and Mechanism Target Effects Reference Transgene, Strain, Frequency Age) 30: Wild-type mice, Anti-VEGF antibody Both treatments act to VEGFA 3.25 and 32.5 pM, IV, single dose Both treatments induced 17 ccc CD1, age and mouse sFlt-1/ reduce circulating VEGF proteinuria by 3 h after – ccc not specified Fc fusion protein administration, which resolved

2019 , (soluble VEGFR1) after 24 h. Both treatments resulted in GEnC hypertrophy and detachment from the basement membrane, starting at 3 h post- treatment and persisting to 24 h Wild-type mice, Antibody to Reduction of circulating VEGFA 100 ml/dose, IP, single dose Decreased glomerular number 21 strain not recombinant VEGFA on days 0, 2, 4, and 5 after birth and formation of abnormal specified, human VEGF165 glomeruli with poor cellularity neonatal and increased ECM. No significant changes in any nonglomerular vessels Wistar Kyoto rats Mouse sFlt-1 Soluble decoy receptor for Soluble VEGFR1 500 mg, IM, 3 d before and 2 wk Accelerated renal failure, 35 treated with plasmid VEGFR1. Reduction of after injection of antiglomerular proteinuria, interstitial antiglomerular circulating VEGFA basement membrane Ab fibrosis, endothelial cell loss. basement and downregulation of membrane Ab, Nephrin in a model of rat 12 wk crescentic GN Wild-type mice, Axitinib Small molecule multitargeted VEGFR2, VEGFR1, 25 mg/kg, IP, twice daily for 7, Reduction in peritubular 51 C57BL/6, (AG-013736) TKI against VEGFR1–3, c-KIT, VEGFR3, c-KIT, 14 or 21 d capillary density by 30% 9–13 wk old and PDGFR PDGFR For dose-response studies: and glomerular capillary 1, 10, or 100 mg/kg, oral by 10% after 21 d of gavage, twice daily for 7 d treatment. Dose dependent

EFIhbto n Nephrotoxicity and Inhibition VEGF increase in proteinuria. Reduced glomerular capillary fenestrations. No increase in serum creatinine Wild-type mice, Ad-sVEGFR1 Adenoviral vector that expresses Soluble VEGFR1 13109 plaque-forming units, No significant reduction in 51 www.jasn.org C57BL/6, the extracellular domain of tail vein, once peritubular capillary or 9–13 wk old murine VEGFR1. Acts as soluble glomerular capillary decoy receptor for VEGF density. Reduced glomerular capillary fenestrations and

increase proteinuria REVIEW after 14 d 5 6

Table 2. Continued REVIEW

Animal Model ora fteAeia oit fNephrology of Society American the of Journal (Model/ Dose, Route and Drug Mechanism Target Effects Reference Transgene, Strain, Frequency www.jasn.org Age) BALB/c(Bicc1/Bicc1) Tesevatinib TKI including EGFR, HER2/ErbB2, VEGFR2, HER2, 7.5 and 15 mg/kg, IP, Dose-dependent reduction in 48 BPK model c-Src, and VEGFR2 EGFR2, ERBB2 daily postnatal day 4–21 whole kidney size, total kidney (murine weight; altered renal and liver phenocopy of morphology ARPKD) and BALB/c wild-type controls, age not specified PCK rat model Tesevatinib TKI including EGFR, VEGFR2, HER2, 7.5 and 15 mg/kg, oral gavage, Dose-dependent reduction in 48 (orthologous HER2/ErbB2, c-Src, EGFR2, ERBB2 daily for 60 d (from whole kidney size, total kidney model of and VEGFR2 postnatal day 30–90) weight; altered renal and liver human ARPKD) morphology and Sprague– Dawley wild type as control, age not specified UUO model and Nintedanib A multitargeted TKI that blocks PDGFR, VEGFR, 50 mg/kg, oral gavage, Attenuated renal fibrosis, inhibited 49 folic acid (BIBF11220) PDGFR, VEGFR, FGFR, and Src FGFR, SRC administered starting on day of activation of renal interstitial nephropathy family kinases UUO and then daily for 7 d fibroblasts, and suppressed models in expression of proinflammatory male wild-type cytokines after UUO C57BL/6 mice, 6–8wk db/db and db/m dRK6 (a D-amino An arginine-rich anti-VEGF VEGFA 50 mg, SC, three times per Both short-term and long-term 36 male C57BL/6 acid derivative hexapeptide that binds with week starting at 8 wk of age and treatment had decreased mice, 6 wk of RK6) VEGF-A, and blocks the lasting until 12 wk (short-term) creatinine clearance compared interaction between VEGFA and 20 wk (long-term) with control db/db mice. Long- (mainly VEGF165 and VEGF121 ) term treatment also mScNephrol Soc Am J and the VEGFRs exacerbated albuminuria, mesangial matrix expansion, and glomerulomegaly as compared with vehicle-treated db/db and short-term dRK6-

30: treated db/db mice 50

ccc Perinatal wild-type DC101 mAb against the extracellular VEGFR2 0.08 mg/dose, IP, on postnatal Large renal cysts, impaired mice (exact portion of the VEGFR2 days 2 and 4 glomerulogenesis – ccc age not specified) (hypocellular), and increased ,2019 albuminuria by 3 wk of age www.jasn.org REVIEW

called multitargeted TKIs, and have differ- ent effects depending on their specificity. 52 TKIs targeting VEGFR in clinical use as Reference chemotherapeutics include sunitinib, pazopanib, sorafenib, and axitinib, and brin

fi differ because of their distinctive pharma- codynamic properties (Figure 1iv). The varying effects of TKIs in various murine models that have used these agents across a spectrum of kidney diseases reflect Effects the wide range of action and targets of TKIs (Table 2). In the bpk mouse model for au- tosomal recessive PKD, intraperitoneal deposits in glomerular capillaries and small arteries hypertension and proteinuria. Intermediate and high doses were associated with endothelial swelling. High-dose only was associated with

All doses associated with injection of tesevatinib (a multikinase in- hibitor targeting EGF receptor, HER2, and VEGFR) from postnatal days 4–21 attenu- ated cyst formation in the kidney and liver rosine-protein kinase Kit; PDGFR, PDGFR receptor; ARPKD,

broblast growth factorptor; rece SC, subcutaneous. 48

fi and reduced total kidney weight. Treat- ment with the multitargeted TKI ninteda- nib attenuated renal fibrosis in wild-type mice subjected to unilateral ureteral ob- Frequency struction and folic acid nephropathy.49 Dose, Route and In contrast to the renal benefit seen

(14 mg/kg) or high (26.7 mg/kg), oral gavage, once daily for 8 d with multitargeted TKIs, inhibition of

Low dose (7 mg/kg), intermediate VEGFR2 alone with an antibody resulted in spontaneous renal cyst development when given perinatally in mice,50 and giv-

, , ing 10-week-old mice axitinib (a TKI tar- geting primarily VEFGR1, VEGFR2, and VEGFR3) resulted in loss of glomerular VEGFR3, PDGFR a PDGFR b FMS-like tyrosine kinase-3 capillary fenestrations as well as protein- VEGFR1, VEGFR2, uria.51 Similarly, Wistar Kyoto rats given sunitinib, which mainly targets PDGF and VEGF receptors, exhibited proteinuria, , hypertension, and endothelial injury.52

l; ECM, ; Ab, antibody; IM, intramuscular; c-KIT, ty A review of 72 RCTs using TKIs targeting VEGFR1/VEGFR2 found that these drugs, like monoclonal anti-VEGF antibody thera- and PDGFR b

a pies, were associated with an increased risk of hypertension. Among patients receiving the TKIs, the total incidence of all-grade or high- VEGFR1, VEGFR2, and VEGFR3, and FMS-like tyrosine kinase-3 PDGFR

A multitargeted TKI including grade hypertension was 23.0% and 4.4%, respectively; sunitinib, pazopanib, cabozan- tinib, , , , , and sorafenib were associated with the highest risk.53 A separate study in- Drug Mechanism Target volving 1120 patients treated with TKIs observed a rapid and significant increase in Sunitinib systolic and diastolic BP after initiation of therapy, with a median onset of 29 days after first dose. Risk factors for treatment-in- Continued ducedhypertensioninthatcohortwerepre- ed Age) fi 300 g, age existing hypertension, body mass index – (Model/ . . 54

Animal Model 25, and age 60 years. Similarly, the rats 280 not speci Transgene, Strain, Male Wistar Kyoto Table 2. IV, intravenous; GEnC, glomerular endothelial cells; IP, intraperitonea autosomal-recessive polycystic kidney disease; PCK, polycystic kidney; UUO, unilateral ureteric obstruction; EGFR, EGF receptor; FGFR, incidenceofall-gradeandhigh-grade

J Am Soc Nephrol 30: ccc–ccc, 2019 VEGF Inhibition and Nephrotoxicity 7 REVIEW www.jasn.org

Table 3. Clinical manifestations of VEGF inhibition Renal Manifestations Druga Class References (Level of Evidence) Bevacizumab mAb against VEGFA Proteinuria 21%–62% (level 1), all-gradeb hypertension 23.6% and high-gradec 8,9,34,37,38 hypertension 7.9% (level 1), renal TMA (levels 6 and 7), MCD/cFSGS-like glomerulopathy (level 6), ABMR-intraocular (level 7) Ranibizumab mAb against VEGFA Systemic and renal TMA (level 7), proteinuria (level 7), ABMR-intraocular (level 7) 46,47 (intraocular) Aflibercept Recombinant fusion protein All-gradeb hypertension 42.4% and high-gradec hypertension 17.4% (level 1), 9,40 proteinuria, TMA (level 6), ABMR-intraocular (level 7) Ramucirumab mAb against VEGFR2 All-gradeb hypertension 21% (level 1), proteinuria 9% (level 1) 39 Sunitinib Multitargeted TKI All-gradeb hypertension 14.9% (level 1), MCD/cFSGS-like glomerulopathy (level 6), 9,53,56 nephrotic syndrome (level 7) Pazopanib Multitargeted TKI All-gradeb hypertension 47% (level 1), all-graded proteinuria 13.5% and high-grade 53,55 proteinuria 2.2% (level 1) Sorafenib Multitargeted TKI All-gradeb hypertension 18.1% (level 1), MCD/cFSGS-like glomerulopathy (level 6), 9,53,55 TMA (level 6), all-graded proteinuria 11.6% and high-gradee proteinuria 0.9% (level 1) Multitargeted TKI All-gradeb hypertension 32.7% (level 1) 53 Vandetanib Multitargeted TKI All-gradeb hypertension 17.3% (level 1), all-graded proteinuria 10.0% and 53,55 high-gradee proteinuria 0% (level 2) Motesanib Multitargeted TKI All-gradeb hypertension 26.1% (level 1) 53 Cediranib Multitargeted TKI All-gradeb hypertension 42.5% (level 1), all-graded proteinuria 37.8% and high- 53,55 gradee proteinuria 3.9% (level 1) Axitinib Multitargeted TKI All-gradeb hypertension 27.1% (level 1), all-graded proteinuria 20.2% and high- 53,55 gradee proteinuria 4.6% (level 1) Regorafenib Multitargeted TKI All-gradeb hypertension 32.4% (level 1), all-graded proteinuria 7.0% and high- 53,55 gradee proteinuria 1.4% (level 2) Multitargeted TKI All-graded proteinuria 9.6% and high-gradee proteinuria 1.5% (level 1) 55 Linifanib Multitargeted TKI All-graded proteinuria 27.3% and high-gradee proteinuria 6.8% (level 1) 55 Multitargeted TKI Nephrotic syndrome (level 7) 56 Multitargeted TKI Nephrotic syndrome (level 7) 56 Multitargeted TKI Nephrotic syndrome (level 7) 56 Vemurafenib BRAF inhibitor AKI, acute tubular necrosis (level 7) 73 Dabrafenib BRAF inhibitor Nephrotic syndrome with severe podocyte and endothelial cell injury 26 when used in combination with trametinib (level 7) Trametinib MEK inhibitor Nephrotic syndrome with severe podocyte and endothelial cell injury when used in 26 combination with dabrafenib (level 7) Levels of evidence: level 1: systematic review or meta-analysis of RCTs; level 2: one well designed RCT; level 3: one controlled trial without randomization; level 4: case–control or cohort studies; level 5: systematic review of descriptive and qualitative studies; level 6: one descriptive or qualitative study; level 7: case series or case report. MCD/cFSGS-like, minimal change disease and/or collapsing FSGS; ABMR, Antibody Medicated Rejection. aAll drug delivery routes are systemic unless otherwise specified. bAll-grade hypertension: grades 1–4. Grade 1: asymptomatic transient increase in systolic BP/diastolic BP .20 mm Hg or ,150/100 not requiring therapy; grade 2: recurrent or persistent or symptomatic increase in systolic BP/diastolic BP .20 mm Hg or .150/100, not requiring therapy; grade 3: 4equires therapy or more intensive therapy than previous; grade 4: hypertensive crisis. cHigh-grade hypertension: combined grades 3 and 4. dAll-grade proteinuria: grades 1–5 (National Cancer Institute toxicity grading criteria version 2 and 3 for proteinuria). Grade 1: dipstick 11 or 0.15–1.0g/24h; grade 2: dipstick 21 to 31 or 1.0–3.5 g/24 h; grade 3: dipstick 41 or .3.5 g/24 h; grade 4: nephrotic syndrome; grade 5: death. eHigh-grade proteinuria: grades 3–5. proteinuria across 33 clinical trials of patients collapsing FSGS9 (Figure 2). In addition, a biopsy sample from the fourth patient with solid tumors treated with VEGFR TKIs case series of four pediatric patients presen- showed histology and electron microscopy was 18.7% and 2.4%, respectively.55 ted by Ruebner et al. reported that the ad- findings were consistent with MCD56;an- In contrast to renal biopsy specimens ministration of TKIs (imatinib, dasatinib, other patient had laboratory evidence con- from patients treated with anti-VEGF quizartinib, and sunitinib) produced severe sistent with TMA in addition to nephrotic mAbs or VEGF trap, biopsy specimens for nephrotic syndrome with marked albumin- syndrome.56 the majority of the patients receiving TKIs uria, edema, and decreased serum albumin. Some evidence suggests that the increase exhibited podocytopathies, including Although full evaluation was limited by a inpodocyteinjurysubsequenttoTKIsmight minimal change disease (MCD) and lack of biopsy in three of the patients, the be mediated by tyrosine phosphorylation of

8 Journal of the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc,2019 www.jasn.org REVIEW

AB Anti-VEGF ligand Tyrosine Kinase Inhibitor

VEGFA ReLA

VEGFA VEGFR2 VEGFR2

VEGFA C-mip

ReLA ReLA ReLA C-mip Podocyte Podocyte C-mip

VEGFA

ReLA ReLA ReLA

Glomerular Endothelial Cell Glomerular Endothelial Cell

C3b C5b-9

VEGFR2 VEGFA CFH CFH VEGFR2

Minimal Change Disease/Focal Thrombotic Microangiopathy Segmental Glomerulosclerosis • Increased complement activation • Increased c-mip podocyte expression • Increased NFκB signaling in both • Unknown effects on complement regulation podocytes and endothelial cells

Figure 2. Inhibition of VEGFA-VEGFR2 signaling has differential downstream effects, depending on the therapeutic target. (A) During treatment with anti-VEGF ligand, podocyte secreted VEGFA is sequestered and does not bind to either podocyte or endothelial VEGFR2 (large X). This leads to increased NF-kB signaling and RelA translocation to the nucleus in both glomerular endothelial cells as well as podocytes. CFH is downregulated on glomerular endothelial cells, which leads to increased complement activation. (B) Treatment with TKIs allows VEGFA to bind to glomerular endothelial and podocyte VEGFR2, but inhibits downstream signaling (small x's). This induces RelA retention in the cytoplasm and c-mip overexpression in the podocyte, leading to alterations in the cytoskeleton and nephrotic syndrome. The effects of TKIs on complement activation remain unclear.

57 nephrin. Simons et al. showed that tyro- of Veg fa164 in transgenic mice not only re- VEGF trap) also attempted to elucidate sine phosphorylation of nephrin, an essen- sulted in expression of VEGFR2 protein in mechanisms behind the two distinct pat- tial slit-diaphragm protein in podocytes, is podocytes, but the VEGFR2 was also tyro- terns of glomerular injury, TMA and critical to maintain the integrity of the fil- sine phosphorylated and colocalized with MCD/FSGS,afterVEGFinhibition(Figure tration barrier. This was demonstrated in a nephrin, effects not seen in wild-type 2).61 Immunostaining revealed that MCD/ murine model, in which an injection of an- mice.28 In the same study, the re- FSGS lesions are associated with increased tibody against nephrin-containing lipid rafts searchers used whole kidney lysates to glomerular C-Maf–inducing protein caused effacement of podocyte foot process- coimmunoprecipitate VEGFR2 and (c-mip), which was not seen in TMA pa- es.57 Furthermore, New et al.58 demon- nephrin, demonstrating that the two tients. In TMA biopsy specimens but not strated in a knock-in murine model that physically interact in vivo. This suggests in MCD/FSGS biopsy specimens, glomer- blocked tyrosine phosphorylation of neph- that when VEGFA availability increases, ular RelA, a subunit of the transcription rin that the mice developed proteinuria with VEGFR2 is expressed in podocytes in factor NF-kB, was significantly upregula- podocyte effacement, emphasizing the im- addition to endothelial cells, and can ted, reflecting increased NF-kBactivity.61 portance of direct nephrin phosphorylation be phosphorylated by podocyte-derived The authors showed that the massively on the maintenance of podocyte morphol- VEGFA.28 These changes were associ- upregulated RelA seen in TMA directly ogy.59,60 Interestingly, administration of the ated with glomerulomegaly, mesangial suppresses c-mip activity by binding to TKI sunitinib for eight consecutive days in expansion, basement membrane thick- its promotor.61 This association of c-mip normotensive Wistar Kyoto rats resulted ening, and foot process effacement.28 overexpression and direct podocyte in- in a dose-dependent decrease in expres- Molecular studies of biopsied kidney jury has been characterized previously in sion of Nephrin mRNA; however, protein samplesfrom29patientswithsolidtumors human, murine, and in vitro studies.62 levels or phosphorylation status were not after they were treated with either a TKI In all, the diverse renal effects of the quantified.52 Further, in another study, in- (sunitinib,axitinib,orsorafenib)oradirect various TKIs can be ascribed to their mul- duction of podocyte-specific overexpression anti-VEGF therapy (bevacizumab or titargeted design. Specific mechanisms of

J Am Soc Nephrol 30: ccc–ccc, 2019 VEGF Inhibition and Nephrotoxicity 9 REVIEW www.jasn.org directpodocyteinjuryafter treatmentwith Y1175 is the major VEGFA-dependent adverse renal events have emerged so far.74 TKIs can be attributed to modulation of autophosphorylation site on VEGFR2. Still, the potential nephrotoxicity associated NF-kB activity and c-mip expression, as Furthermore, mutations introduced at with the inhibition of key proteins in the well as nephrin phosphorylation and in- the Y1175 phosphorylation domain on RAS-RAF-MEK-MAPK-ERK1/2 pathway teraction with VEGFR2, but require vali- VEGFR2 resulted in loss of the ability to demonstrates a need to further explore dation in future studies. tyrosine phosphorylate phospholipase downstream effectors of extracellular recep- C-g,aswellasasignificant reduction in tor activation that are specifictothetumor,as MAPK phosphorylation and a corre- well as minimize nephrotoxicity. VEGF SIGNALING PATHWAYS sponding reduction in DNA synthe- sis. These effects suggest that Y1175 Toget a full picture of the nephrotoxicity plays a critical role in MAPK signal ENOS SIGNALING associated with inhibition of VEGF transduction.68 signaling, it is critical to gain an under- The importance of autophosphoryla- Some investigators have postulateda reduc- standing of the downstream mecha- tion of VEGFR2 was also subsequently tion in the vasodilator eNOS as a potential nisms and regulatory cascades initiated demonstrated by Sakurai et al.69 who mechanism underlying the hypertension by VEGFR2 activation. Key downstream used a constitutive knock-in mouse and endothelial injury seen subsequent to signaling pathways include MAPK/ model with phenylalanine residues pharmacologicVEGFinhibition.Micewith ERK1/2, endothelial nitric oxide syn- 2 2 substituting for tyrosine residues in the eNOS deficiency (Nos3 / ) exhibit endo- thase (eNOS), and mammalian target Veg fr2 gene—a change that was embry- thelial injury in the form of increased plate- of rapamycin (mTOR). onically lethal because of loss of blood let aggregation,75 leukocyte adhesion,76 vessel differentiation. Furthermore, the propensity toward thrombosis,76 and RAF/MAPK/ERK SIGNALING critical nature of VEGF-ERK signaling hypertension.77 Downstream of VEGFA- was underscored in a study of a cohort VEGFR2 signaling in human umbilical One pathway that has been extensively of patients with hepatocellular carci- vein cells78 and glomerular endothe- studied is VEGF-induced ERK1/2 signal- noma that found that tumors with high- lial cells, phosphorylation of eNOS at ing, which serves to regulate endothelial est expression of phospho-ERK were Ser1177induceseNOS in a time-dependent differentiation and proliferation.63 In the more responsive to VEGF inhibition manner, leading to nitric oxide genera- kidney, MAPK/ERK signaling has diverse via TKI.70 tion.79 Furthermore, the angiogenic effects. Parietal epithelial cells in experi- Therapeutic inhibitors that target various effects of VEGF-VEGFR2 are mitigated mental FSGS64 show increased ERK1/2 portions of the MAPK/ERK are currently in in the absence of eNOS.80,81 Evidence sup- activation, and it is also associated with development as novel . porting the role of VEGF in the hyperten- increased podocyte in diabetic Some agents, such as vemurafenib and dabra- sive response comes from Tang et al.82 who conditions.65 In contrast to its association fenib, specifically target B-Raf, an upstream found that a single dose of the selective with pathologic consequences in renal ep- component of the intracellular MAPK/ERK VEGFR2 inhibitor SU5416 significantly ithelial cells, in glomerular endothelial intracellular pathway (Figure 1v). Mutated downregulated lung eNOS expression in cells, MAPK/ERK signaling is required B-Raf, as is found in various cancers,71 results 3-day-oldratsandinducedpulmonaryhy- for the protective effects of in persistently elevated ERK phosphorylation. pertension and right ventricular hypertro- 1 against endoplasmic reticulum stress.66 Clinically apparent renal toxicity with the use phy; these effects were attenuated with In cultured glomerular endothelial cells, of B-Raf inhibitors most often occurs in the inhaled nitric oxide therapy. There are re- ERK1/2 inhibition completely suppressed tubulointerstitial compartment, manifests as ports of the TKIs dasatinib and VEGF-mediated proliferation, but migra- AKI, and is more prevalent in males, accord- inducing pulmonary arterial hypertension tion was not affected.67 ing to a recent review (Table 3).72 Specifically, after their use in patients,83 and ponatinib Outside of the kidney, VEGF induces vemurafenib treatment in eight patients with administered to cultured human aortic en- ERK1/2phosphorylationinatime-andcon- malignant melanoma resulted in severe AKI; dothelial cells has been similarly associated centration-dependent manner and is re- the single biopsy performed revealed acute tu- with decreased NOS3 expression.84 sponsible for its effects on endothelial cell bular necrosis.73 Interestingly, treatment with In light of eNOS’s essential role of in reg- hyperpermeability.20 This VEGF-mediated the combination of dabrafenib and the MEK ulating vascular endothelial integrity ERK1/2 signaling is dependent on the up- inhibitor trametinib resulted in severe ne- through VEGF signaling, it is important to stream mediators Raf-1 and MEK, as inhi- phrotic syndrome with podocyte effacement consider the contribution of other key en- bition of either attenuated VEGF-induced and glomerular endothelial cell injury on bi- dothelial regulatory genes in this pathway. endothelial cell hyperpermeability.20 opsy that was reversible upon therapy discon- Krüppel-like factor 2 (KLF2) and Krüppel- In addition, Takahashi et al. showed tinuation.26 Direct ERK1/2 inhibitors are also like factor 4 (KLF4) are well known zinc- the importance of the VEGFR2/MAPK/ in development. A recently completed phase 1 finger transcription factors that regulate ERK downstream pathway in cultured trial of the first of these agents to emerge from anti-inflammatory85,86 and antithrom- endothelial cells by determining that preclinical studies, ulixertinib, noted that no botic87 pathways in the endothelium.

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KLF2 induces both eNOS expression and glomerular endothelial damage in diabetic signaling, as both ACE inhibitors and angio- activity in endothelial cells by regulating mice97 suggests that modulation of the tensin receptor blockers have been shown to its promoter,88 and KLF4 has also been mTOR pathway in endothelial cells may increase kidney eNOS levels after ischemia- demonstrated to positively regulate eNOS also result in glomerular disease. reperfusion injury.102 expression.89 Interestingly, these two tran- Proteinuria is the main renal effect sub- Guidelines published in 2010, on the ba- scription factors appear to have discrepant sequenttomTORdownregulationinmurine sis of recommendations to that National effects on VEGFA-VEGFR2 signaling: KLF2 models of podocyte injury, as well as the pri- Cancer Institute’s Investigational Drug overexpression in human umbilical vein maryrenalmanifestationobservedinpatients Steering Committee, advise conducting cells inhibited VEGFR2 mRNA and protein after mTOR inhibition. The reported inci- and documenting a formal risk assessment expression, as well as promoter activity, sug- dence of proteinuria varies widely, between of cardiovascular complications before ini- gesting an antiangiogenic effect,90 whereas 3% and 36% after everolimus therapy.98 In- tiation of VEGF inhibition, followed by ac- KLF4 has been demonstrated to positively terestingly, the mTOR inhibitor tive BP monitoring during VEGF inhibition regulate VEGFA expression.91 Further ex- has been associated with de novo TMA in therapy, with a treatment goal of BP ,140/ ploration of these signaling pathways is war- kidney transplant recipients that developed 90 mm Hg.103 The addition of antihyper- ranted to elucidate the mechanisms by in the absence of inhibitors, and tensive agents when BP remains above goal which VEGF inhibition contributes to the in one case series, sirolimus correlated with a is recommended, with some clinical data nephrotoxicity observed in clinical practice. significant reduction in glomerular VEGF suggesting an added benefitofACEinhibi- proteinexpressiondetectedbyimmunos- tion over other classes.104 Temporarily dis- taining.99 This observation was explored fur- continuing VEGF inhibition therapy or MTOR SIGNALING ther in a murine model, in which mice with dose reduction might be necessary if BP podocyte-specificdeletionofmTor devel- control is not possible.103 Another important downstream RTK tar- oped significant proteinuria and ESRD by Microalbuminuria often accompanies hy- get, mTOR, is part of the phosphatidylino- 5weeksofage.100 In this model, VEGFA pertension,38 and first-line therapy is gener- sitide 3-kinase/AKT signal transduction levels were not reduced until the mice were ally renin-angiotensin-aldosterone system pathway (Figure 1vi). Inhibition of mTOR 3 weeks old, after the initiation of disease, inhibition, as this has shown some success signaling blocks VEGF-mediated angio- suggesting multiple pathways are likely in mTOR antagonist–associated albumin- genesis and endothelial cell proliferation responsible for disease induction and pro- uria after kidney transplantation.105 Albu- at two different levels: by reducing VEGF gression.100 Accordingly, the mechanisms minuria should be quantified before synthesis and secretion, and by reducing behind renal toxicities related to mTOR in- initiating therapy, and significant protein- VEGFR2-mediated signaling.92 The mech- hibition have been postulated to be related uria (.2 g in 24 hours) is cause for anism responsible for the decreased VEGF to a decrease in VEGF signaling, as well as to discontinuation of therapy per 2013 guide- production is not fully understood, but it disruption of the autophagic pathway.100 lines,106 as is nephrotic syndrome or TMA. does not appear to be due to modulation of In the cases of nephrotic-range albumin- upstreamgenessuchasHIF-1a or uria, hematuria, or biochemical evidence TGF-b.92 Activation of mTOR occurs in CLINICAL CONSIDERATIONS FOR of impaired kidney function, kidney biopsy conditions such as tuberous sclerosis and MINIMIZING NEPHROTOXICITY should be pursued, as glomerular diseases Peutz–Jeghers syndrome, which feature ab- associated with VEGF inhibition vary. errant cell proliferation and a tendency to Managing the various renal toxicities In all, close monitoring during therapy develop malignancies,93 and mTOR inhibi- associated with VEGF inhibition will and a thorough assessment of renal tors such as temsirolimus, ridaforolimus, remain an important area of ongoing re- function before initiation, including mi- and everolimus have shown promising re- search because of the wide use of these croalbuminuria, hematuria, and serum sults in the treatment of several cancers.94 agents and the steady development of creatinine, should be performed in all pa- In the kidney, several studies have out- novel therapeutics targeting this pathway tients receiving VEGF inhibition therapy. lined the importance of mTOR in the main- in cancer. Experimental data from rats tenance of podocyte integrity, primarily treated for 5 days with the TKI sunitinib through regulation of autophagy,95 as dele- demonstrated that coadministration of CONCLUSIONS AND FUTURE tion of either of its two functional complexes sunitinib with either the angiotensin- PERSPECTIVES (mTOR complex 1 or mTOR complex 2) converting enzyme (ACE) inhibitor cap- resulted in severe glomerulosclerosis.96 Al- topril or the phosphodiesterase type 5 PharmacologicinhibitionofVEGFsignaling though mTOR’s role in podocytes has been inhibitor sildenafil reduced proteinuria and its downstream pathways is a more extensively studied, the mTOR auto- andhistologicevidenceofendothelialinjury, common therapeutic strategy in oncology, phagic pathway has a role in endothelial cells whereas neither had an effect on sunitinib- but associated nephrotoxicities remain a as well. The finding that endothelial-specific induced hypertension.101 The mechanism concern. Although adverse effects such as knockdown of Atg5, which encodes a key behind these renoprotective effects is unde- hypertension,proteinuria,andTMAare autophagic vesicle protein, exacerbated termined, but it might involve eNOS well described in both experimental and

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