Urinalysis Negative for Blood or Protein

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2 Case hx: 26 y.o. F, immigrated from Korea as infant, healthy until Sep Low grade fever, rec. rash Urticaria like, myalgias Improved with Prednisone, relapsed when dose tapered Jan admitted to community hospital: Sore throat, pleuritic chest pain and abdominal pain. High D-dimer-> CT-PE negative Jan 5 th Abdominal pain with small free peritoneal fluid-> exploratory laparotomy -negative for any pathology discharged home

3 Case hx: Jan 8 th - Lt. eye; painless vision loss- suspected central retinal vein occlusion seen by ophthalmology as outpatient Jan 16 th -Re-admission for investigation- seen by Rheumatology, Dx of Pericarditis, suspected Adult onset Still s disease- started on Prednisone 40mg OD

4 Labratory results: Hb-119, PLT 335, WBC 21,700 Cr- 72 (0.8 US units) Urinalysis Negative for Blood or Protein C3-1.1 (normal) C (normal) CRP-185, ESR-50 (elevated) ANA, ANCA, RF, Lupus anticoagulant- all negative HBV, HCV, HIV, VDRL, Toxoplasmosis- Neg Immunity against CMV and EBV with no sign of recent infection Ferritin >3082 (high)

5 Case hx: Feb2nd: GP visit for increase SOB Cr-648 (7.3), Urea 57.7, K-4.8, HCO3-17 Urinalysis: Protein >3gr/L, +4 RBC, on microscopy heme granular, granular and hyaline casts, No cellular casts LDH-1480 (v. high), PLT-67, HB-83 Fragments were seen on blood film Low C3 Decreased LOC-> MRI non specific changes, consistent with cerebritis transferred to our hospital (SMH) PLEX (plasmapheresis with exchange) and HD (hemodialysis) initiated

6 Thrombotic Microangiopathies Microangiopathic hemolytic anemia, thrombocytopenia and organ injury Thrombi in capillaries and arterioles Glomerular microvasculature is particularly susceptible

7 How can we put the story together?

8 Objectives Revisit the classification of TMAs in the molecular era role of VEGF signaling Explore why the kidney (glomerulus) is particularly susceptible to TMA Understand crosstalk exists between pathways responsible for TMA

9 Thrombotic Microangiopathy TTP HUS D+HUS (typical HUS) Atypical HUS Other TMAs HIV, cancer, drugs, connective tissue disease, transplant rejection,idiopathic HELLP, preeclampsia, anti-vegf Malignant hypertension, antiphospholipid Adapted from: Coppo & Veyradier, Cardiovascular & Hematologic Disorders, 2009, 9: 36-50

10 ADAMTS 13 and TTP (thrombotic thrombocytopenic purpura) Moake, JCI, 1986; David Ginsburg and Tsai., Nature, 2001

11 Hemolytic Uremic Syndrome Noris & Remuzzi, review, NEJM, Oct. 2009

12 Typical HUS (diarrhea associated HUS; D+HUS) Shiga-toxin (STEC e.g. E.coli 0157:H7) Most common cause of HUS Pediatric population Mortality or ESRD: 12%; longterm renal sequelae 25% Shiga toxin enters systemic circ n, binds Gb3 on glomerular endothelial cells platelet clots

13 HUS Outbreak in Germany, people, 845 cases of HUS, 54 fatalities Older age (median age 42 y.o.); females>males; longer incubation period; higher incidence of HUS EAEC (E.coli104:H4) that had acquired shigatoxin gene supervirulent

14 HUS Outbreak in Germany, people, 845 cases of HUS, 54 fatalities Older age (median age 42 y.o.); females>males; longer incubation period; higher incidence of HUS EAEC that had acquired shigatoxin gene supervirulent

15 Atypical HUS (ahus) By definition not caused by shigatoxin Less common 10% of cases Heritable and sporadic cases Abnormalities in complement regulation Low C3 is helpful (*may not be present) Worse outcome: mortality -25% and ESRD in 50%

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17 ahus and Complement Regulation Factor H deficiency or autoantibodies) Factor H-related proteins (1 to 5) Factor I Factor B MCP (CD46) Complement C3 Thrombomodulin Noris & Remuzzi, review, NEJM, Oct. 2009

18 ahus It s not all about complement!

19 O z DGKE- atypical HUS

20 Why can patients with mutations live their whole life without TMA?

21 Why can patients with mutations live their whole life without TMA? 2 hit hypothesis Need another trigger

22 Thrombotic Microangiopathy TTP (low ADAMTS13) ADAMTS13 mutations (deficiency) Autoimmune (autoantibodies) (HIV, pregancy, antiplatelet agents, cancer, idiopathic etc.) HUS (normal ADAMTS13) HUS & enteropathogenic bacteria (Shiga-toxin; D+HUS) Atypical HUS & mutations or defects in complement pathway (Factor H, MCP, Factor B, I, FHR1 & Factor H, thrombomodulin) Strep pneumoniae Cobalamin deficiency DGKE Other TMAs HIV, cancer, drugs, connective tissue disease, transplant rejection, post-transplant rejection idiopathic HELLP, preeclampsia, anti-vegf agents Malignant hypertension, antiphospholipid Adapted from: Coppo & Veyradier, Cardiovascular & Hematologic Disorders, 2009, 9: 36-50

23 Why is the kidney (glomerulus) particularly susceptible to thrombotic injury?

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27 Deen JCI 2004 SD

28 VEGF-A

29 VEGF-A

30 VEGF plays a major role in protecting the glomerular microvasculature from thrombi

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32 Patients on VEGF blockers: Incidence of proteinuria % Nephrotic syndrome has been seen in approx. 1-2% of patients (7% in RCC trials) Hypertension - up to 36% (Bevacizumab) (Up to 43% in TKI studies) Zhu X., Parikh, Meta-analysis, Am J Kidney Dis, 2007

33 Patients and anti-vegf drugs Three single publications of TMA in patients: VEGF trap (Izzedine H et al., Nephrol Dial Transplant, 2007) Bevacizumab/Sunitinib (Frangie C., et al., Lancet, 2007) Sunitinib (Levey et al., NDT, 2008)

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35 Patient biopsy - anti-vegf therapy N = 6

36 Summary of Patients 6 patients Variety of cancers - lung, ovarian, renal cell Dose of Bevacizumab (7.5 mg/kg - 15 mg/kg) Sub-nephrotic to nephrotic range proteinuria Renal function deterioration - variable 5/6 improved off Bevacizumab (1 died) 1/6 had schistocytes and thrombocytopenia

37 Anti-VEGF and TMA Kidney biopsy, single center, 100 patients after anti-vegf 73% TMA; ~ ½ are renal-restricted 27% MCD, FSGS-like pattern most occurred with tyrosine kinase inhibitors* ~70% proteinuria >1g/24 hour Improvement upon D/C drug; recurrence or worsening if drug continued or restarted Izzedine et al., Medicine, 93(24), Nov 2014

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39 Hypothesis VEGF inhibitors cause proteinuria and glomerular injury (TMA) by local reduction of VEGF ( on target effect )

40 Hypothesis VEGF inhibitors cause proteinuria and glomerular injury (TMA) by local reduction of VEGF ( on target effect )

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43 Proof of principle studies

44 Genetic deletion of VEGF from adult, filtering glomeruli podocin x rtta podocin rtta teto Cre X Exon 3 1 P VEGF-A P 4 teto Cre +Dox = rtta = Dox Only in podocyte: P = loxp exon 1 P exon 4

45 Genetic deletion of VEGF from adult, filtering glomeruli podocin x rtta podocin rtta teto Cre X Exon 3 1 P VEGF-A P 4 teto Cre +Dox = rtta = Dox Only in podocyte: P = loxp exon 1 P exon 4

46 In-situ Control Mutant Prior to Dox induction, podocytes express VEGF, but there is no expression of VEGF after + Dox Nephrin expression at the onset of proteinuria is not changed

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48 Endotheliosis

49 Endotheliosis - Patients

50 Summary - POP studies 100% of mice develop glomerular injury characteristic of TMA VEGF produced by the podocytes IS required by glomerular endothelium in a filtering glomerulus TMA observed in patients on VEGF inhibitors - on-target effect and due to local reduction of VEGF

51 Hypothetical Model of Disruption of VEGF Signaling in Renal Thrombotic Microangiopathy Eremina V et al. N Engl J Med 2008;358:

52 Keep an eye on Systemic and Kidney Toxicity of Intraocular Administration of Vascular Endotheilal Growth Factor Inhibitors AJKD, Volume 57, Issue 5, May cases reported in the literature, additional anecdotal cases

53 Case hx: Jan 8 th - Lt. eye painless vision loss- suspected central retinal vein occlusion

54 Case hx: Jan 8 th - Lt. eye painless vision loss- suspected central retinal vein occlusion Rx: Jan 23 and 29 injection of anti-vegf therapy (Lucentis) into eye (intravitreal)

55 Why would she be susceptible?

56 Two hit hypothesis 3 protective/regulatory pathways important for glomerular microvasculature: ADAMTS13 Complement Regulatory Proteins VEGF- VEGF-receptor Signaling

57 Why would she be susceptible? Active disease (Dx: Still s disease) some complement activation Although C3 initially within normal range, low during TMA Blood sent for genetic sequencing at Hospital for Sick Children, Toronto

58 Mutation in C3 * V. Fremeaux-Bacchi, Blood, (112), 2008;

59 VEGF ADAMTS 13 Complement regulation

60 Podocin and Complement Factor H Control Podocyte VEGF-A KD Keir et al., VEGF Regulates Inhibitory Complement Proteins in the Eye and Kidney, JCI, Jan. 2017

61 Patient History 3 weeks of PLEX platelets, Hgb normalized Discharged home outpatient hemodialysis Presented with PRES (posterior reversible encephalopathy syndrome, fragments, hemolysis)

62 Patient History Rx: PLEX and then Eculizimab (released on emergency basis) after Eculizimab dose, remarkable recovery, discontinued dialysis Discharged from ICU to home

63 Treatments ADAMTS 13 : plasma infusion (deficiency) vs. plasma exchange (autoantibodies) Rituximab- monoclonal antibody to B lymphocytes (inhibit antibody formation) Aptamers ARC1779 (inhibit VWF/platelet interaction) Nanobody ALX-0681 (inhibit VWF/platelet interaction) Recombinant ADAMTS13 ahus complement Plasma infusion vs. plasma exchange (dfcy vs. autoantibodies) Eculizamab (C5 inhibitor), + many other complement inhibitors Recombinant Factor H

64 Treatment: anti-vegf & TMA ANTI-VEGF AGENTS Drug holiday most patients recover Reintroduction or continuation: ¾ will develop more severe TMA (examples of no recurrence when switch drugs) PREECLAMPSIA Delivery of fetus Removal of sflt1 extracorporeal column Thadani et al., Circulation, 124(8), Thadani et al.,jasn, Sept 24, online

65 Pilot Study of Extracorporeal Removal of Soluble Fms-Like Tyrosine Kinase 1 in PreeclampsiaClinical Perspective by Ravi Thadhani, Tuelay Kisner, Henning Hagmann, Verena Bossung, Stefanie Noack, Wiebke Schaarschmidt, Alexander Jank, Angela Kribs, Oliver A. Cornely, Claudia Kreyssig, Linda Hemphill, Alan C. Rigby, Santosh Khedkar, Tom H. Lindner, Peter Mallmann, Holger Stepan, S. Ananth Karumanchi, and Thomas Benzing Circulation Volume 124(8): August 23, 2011 Copyright American Heart Association, Inc. All rights reserved.

66 Objectives Revisit the classification of TMAs in the molecular era role of VEGF signaling Explore why the kidney (glomerulus) is particularly susceptible to TMA Understand crosstalk exists between pathways responsible for TMA

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68 10/23/ Charles Alpers* (Seattle) Ashley Jefferson* (Seattle) Paul Rene de Cotret (Quebec) Howard Hochster (NYU) Joseph Weisstuch (NYU) Laura Barisoni* (NYU) Mark Haas (John Hopkins) Paul Thorner (HSC, U of T) Napoleone Ferrara (Genentech) Hans Peter Gerber (Genentech) U of T, SLRI Andras Nagy Jody Haigh Mouse facility

69 Case #2 - Mrs. F, 72 y.o. female with age related macular degeneration receiving intravitreal injections of anti-vegf (ranibizumab) X 2 years Admitted with Creatinine 3.2 and proteinuria (1.3 g/24 hour) Creatinine 6 months earlier: 1.2

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72 As nephrology consultant you recommend: A) Discontinue anti-vegf (ranibuzimab) injections B) Continue anti-vegf agents and look for alternate cause of kidney disease C) Measure circulating VEGF levels and discontinue anti-vegf agents if levels are low D) Discuss risk-benefit with ophthalmologists and patient

73 As nephrology consultant you recommend: A) Discontinue anti-vegf (ranibuzimab) injections B) Continue anti-vegf agents and look for alternate cause of kidney disease C) Measure circulating VEGF levels and discontinue anti-vegf agents if levels are low D) Discuss risk-benefit with ophthalmologists and patient

74 Tyrosine kinase inhibitors VEGF receptor blockers Minimal change disease or FSGS-like lesions

75 VEGFR1/sFLT1 is Produced by Podocytes Dontscho Kerjaschki, Vienna

76 Loss of VEGFR1/Flt1 from podocytes

77 Jin et al.,sflt1 regulates podocyte cytoskeleton Cell, 2013 Model Nephrin

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79 Specific organ injury in TMA may be explained by: A) Glomerulus is a high flux vascular bed under constant mechanical stress B) Complement activation is high in the glomerular microvasculature C) Specialized features of glomerular endothelium e.g. fenestrations, make it prone to injury D) Organ-specific features and targeted injury of blood vessels determine which tissues are targeted during TMA (e.g. molecular, mechanical, external forces) E) All of the above

80 Case #3 59 y.o. male with Type II DM, DME (diabetic macular edema), Stage 3b CKD

81 As nephrology consultant you recommend: A) Avoid anti-vegf agents and try second line therapy B) Perform baseline Creatinine clearance and 24 hour urine protein measurement arrange f/u in office C) Choose the anti-vegf agent with shortest half-life (Aflibercept < Ranibizumab < Bevacizumab) D) Choose the anti-vegfagent with least affinity to VEGF (Bevacizumab/Ranibizumab < Aflibercept E) B & C

82 Thrombotic Microangiopathy TTP (low ADAMTS13) ADAMTS13 mutations (deficiency) Autoimmune (autoantibodies) (HIV, pregancy, antiplatelet agents, cancer, idiopathic etc.) HUS (normal ADAMTS13) HUS & enteropathogenic bacteria (Shiga-toxin; D+HUS) Atypical HUS & mutations or defects in complement pathway (Factor H, MCP, Factor B, I, FHR1 & Factor H, thrombomodulin) Strep pneumoniae Cobalamin deficiency DGKE Other TMAs HIV, cancer, drugs, connective tissue disease, transplant rejection, post-transplant rejection idiopathic HELLP, preeclampsia, anti-vegf agents Malignant hypertension, antiphospholipid Adapted from: Coppo & Veyradier, Cardiovascular & Hematologic Disorders, 2009, 9: 36-50

83 Changes in maternal circulating sflt-1 levels (pg/ml) (A), protein-creatinine ratio (g/g) (B), and blood pressure (mm Hg) (C) in patient 3 over the course of 23 days of inpatient hospitalization starting at gestational age 27 weeks+4 days. Thadhani R et al. Circulation. 2011;124: Copyright American Heart Association, Inc. All rights reserved.

84 VEGF-A

85 VEGF-A

86 VEGF-A