This case report describes a 63-year-old man who developed acute kidney injury and thrombotic microangiopathy after receiving treatment with the proteasome inhibitor carfilzomib. A kidney biopsy showed features of thrombotic microangiopathy and arteriolar damage. The authors propose that proteasome inhibitors may cause kidney injury by decreasing VEGF production through inhibition of the NF-κB pathway, disrupting normal endothelial cell function and integrity. The patient's kidney function recovered after discontinuing carfilzomib with supportive management. This case adds to evidence that proteasome inhibitors can cause thrombotic microangiopathy through their effects on VEGF signaling.

2. Contributors
• Ahad Lodhi, M.D , Abhishek Kumar, M.D ,
Muhammad U Saqlain, M.D, Manish Suneja
M.D .
• Dept. of Nephrology, Internal Medicine,
University of Iowa Hospitals and Clinics, 200
Hawkins Dr, Iowa City IA 52246

3. • Transparency Declaration:
• None to declare

4. Theme
• Proteasome inhibitors
• Proteasome inhibitors and AKI
• Case report with brief discussion
• Pathogenesis of TMA with proteasome
inhibitors

5. Proteasome Inhibitors

7. • The Ubiquitin proteasome pathway has a key
role in in the targeted destruction of cellular
proteins, including cell cycle regulatory
proteins. This process is central for cell cycle,
function and survival, making proteasome
inhibition a potential target in cancer.

8. Bortezomib
• first inhibitor of proteasome pathway to be
approved in 2003 for clinical use in US
• It is boron molecule that reversibly inhibits
threonine residue of 26S proteasome

9. Bortezomib

10. • Initially approved for the treatment of
myeloma in the post transplant relapse or as a
second line treatment in patients unsuitable
for transplantation
• In 2014 ,also approved in patients who
responded to prior therapy with the same
and relapsed at least six months following
completion
Richardson PG, Barlogie B, Berenson J, et al. A Phase 2 study of bortezomib in relapsed, refractory
myeloma. N Engl J Med. 2003;348:2609–17
Jagannath S, Barlogie B, Berenson J, et al. A phase 2 study of two doses of bortezomib in relapsed or
refractory myeloma. Br J Haematol. 2004;127:165–72

11. Carfilzomib (CFZ)
• Second generation proteasome inhibitor that
was developed to overcome side effects with
Bortezomib, mainly peripheral neuropathy
• It was approved by FDA in 2012 for patients
with MM who had relapsed from and were
refractory to bortezomib and at least one
thalidomide derivative
Herndon TM, Deisseroth A, Kaminskas E, et al: U.S. Food and Drug Administration approval:
Carfilzomib for the treatment of multiple myeloma. Clin Cancer Res 19:4559-4563, 2013
Demo SD, Kirk CJ, Aujay MA, et al: Antitumor activity of PR-171, a novel irreversible inhibitor of the
proteasome. Cancer Res 67:6383-6391, 2007

14. Proteasome Inhibitors and AKI

15. • Few cases, have been reported citing Bortezomib
and Carfilzomib as a cause of AKI
• Underlying mechanism of kidney injury has been
unclear
• Vasoconstriction of renal arteries leading to AKI,
has been hypothesized by Wanchoo et al
• Use of N-acetyl-L-cysteine in a reported patient
partially mitigated the renal injury upon re-
challenge of CFZ
Jhaveri, K.D., et al., Carfilzomib-related acute kidney injury. Clin Adv Hematol Oncol,
2013. 11(9): p. 604-5
Wanchoo, R., et al., Carfilzomib-related acute kidney injury may be prevented by N-
acetyl-L-cysteine. J Oncol Pharm Pract, 2014

16. Bortezomib and AKI
• FDA approval of bortezomib in June 2008
Phase-2 trials CREST and SUMMIT involving
54 and 202 patients, respectively.
• Acute Kidney injury or TMA was not reported
as a drug-related adverse event in these early
trials.
Jagannath S, Barlogie B, Berenson JR, Siegel DS, Irwin D, Richardson PG, Niesvizky R, Alexanian R, Limentani SA,
Alsina M, Esseltine DL, Anderson KC. Br J Haematol. 2008 Nov;143(4):537-40. doi: 10.1111/j.1365-
2141.2008.07359.x. Epub 2008 Sep 6
Jagannath S, Richardson PG, Barlogie B, Berenson JR, Singhal S, Irwin D, Srkalovic G, Schenkein DP, Esseltine DL,
Anderson KC; SUMMIT/CREST Investigators. Haematologica. 2006 Jul;91(7):929-34.

17. Carfilzomib and AKI
• FDA approval of Carfilzomib in July 2012 was
phase-2 trial by Siegel et al
Siegel, D.S., et al., A phase 2 study of single-agent carfilzomib (PX-171-003-A1) in patients with relapsed and
refractory multiple myeloma. Blood, 2012. 120(14): p. 2817-25

18. Siegel, D.S., et al., A phase 2 study of single-agent carfilzomib (PX-171-003-A1)
in patients with relapsed and refractory multiple myeloma. Blood, 2012.
120(14): p. 2817-25
266
Patients
• Total no of
patients in
the study
25% • Inc in Cr,
>1.5
5%
• Cr >3
times the
baseline

19. 526 pts 24.1% inc in Cr
17.7%
treatment
related
It was interesting to note that fever, anemia and
thrombocytopenia was also reported in 30%, 46% and 29% of
patients respectively

22. • There have been four cases of BTZ associated
AKI with evidence of underlying TMA (table 1).
A kidney biopsy was not performed in any of
these cases
• Hobeika et al, recently published a case of a
patient presenting with worsening
hypertension and proteinuria after CFZ
exposure
BMC Nephrol. 2014 Sep 30;15:156. doi: 10.1186/1471-2369-15-156.

23. • Kidney biopsy showed features of TMA and
associated podocytopathy, with foot process
effacement
• This presentation was not associated with
Microangiopathic hemolytic anemia (MAHA)
• Similar pathology has also been described in
TMA associated with VEGF inhibitor exposure

24. Case Report

25. Presentation
• A 63 yo Caucasian male with multiple
myeloma (MM) IgG kappa, ISS stage II, status
post 2 autologous bone marrow transplants
(last one about 6 months prior to this
presentation) was found to have AKI (sCr of
6.3mg/dl) during his routine visit to receive
his second dose of CFZ.

26. Brief HPI
• Two weeks prior to this presentation he was
started on chemotherapy with CFZ
• His sCr was 0.7 mg/dl at this time with no
evidence of proteinuria
• His past medical history was also significant
for hypertension, dyslipidemia, urate
nephrolithiasis

27. HPI Cont..
• He was initially diagnosed with MM about 18
months ago when he had presented with a right
pleural-based mass, which was associated with a
destructive lesion of an adjacent rib
• A FNA of the lesion was performed at that time
which showed sheets of plasma cells consistent
with multiple myeloma
• His kappa to lambda ratio (κ/λ) a year ago was
75.84 and was 1.58 at the time of this
presentation

28. • Worsening lower extremity swelling weight gain
• Decreasing urine output
• No diarrhea, shortness of breath, chest
discomfort, orthostatic symptoms or any
exposure to any nephrotoxins including contrast
agents
• Current medications : acyclovir, dexamethasone,
fluconazole, fludrocortisone, gabapentin,
loratadine, omeprazole, pentamidine inhaled,
propranolol, simvastatin and trazodone

29. EXAM
• Temp 98.6 °F (37C), BP 141/80 mm of Hg (no
evidence of orthostasis), pulse 94 /min and O2
sat 97% on RA
• Physical exam revealed mild diffuse bilateral
wheezing and lower extremity edema up to
knees

30. -Urinalysis was significant for
2+blood and 2+ proteins
without casts or crystals
-Urine microscopy was bland
-Urine sodium was 33mEq/L
(33mmol/L) and urine Cr was
97.1 mg/dl (8583 µmol/L)
with UPR of 1.5
-Antiphospholipid antibodies
and ANCA serologies were
negative
-Serum complements (C 3
and C4) were within normal
range
-Renal ultrasound was
unremarkable without any
evidence of hydronephrosis

31. -14 0 2 4 6 8 10 12 18
Creatinine 0.7 6.3 6.2 5.5 4.9 3.9 3.3 2.9 2.5
Hemoglobin 11.3 5.7 6.8 7.4 8.1 7.3 7.1 8.2 7.4
0
2
4
6
8
10
12
Chart 1

32. 182
176
35 30
34
53
76
96
123
106
196
170
777
727
304
235
180
167 145
121
0
100
200
300
400
500
600
700
800
900
0
20
40
60
80
100
120
140
160
180
200
Chart 2
Platelets LDH
Day of

33. Course
• A presumptive diagnosis of TTP/HUS was made
and empiric plasmapheresis was started
• Patient was not re-challenged with CFZ again
• Plasmapheresis was continued while ADAMTS13
levels were pending
• ADAMTS13 results (more than 50%) were
available on day 10 and thus plasmapheresis was
stopped after 5 sessions.
• Patient never required dialysis and over the next
week, his sCr started to improve with improving
urine out

34. Biopsy
• Patient underwent an ultrasound guided
kidney biopsy one week after presentation,
when platelets were greater than 65000/mm3
• Biopsy revealed glomerular and arteriolar
TMA with mild ATN

36. • His hemoglobin, platelets, LDH and haptoglobin
normalized over the next three weeks
• Patient was treated supportively for the rest of
his stay
• His sCr continued to improve and last known
value 45 days after the initial presentation was
1.8 mg/dl (159 µmol/L)
• Workup for other causes of atypical HUS (e.g.
CFH autoantibodies) was not done, though
normal complement level and improvement in
TMA argues against it.

37. Discussion
• We postulate CFZ as a potential etiology of TMA
• Discontinuation of CFZ along with supportive
management was associated with renal recovery
in our patient
• This patient presented with AKI secondary to
TMA associated with MAHA (elevated LDH, low
haptoglobin and schistocytes on peripheral blood
smear) and thrombocytopenia

38. • Causality is difficult to establish,
• No other obvious cause of AKI including
volume depletion, tumor lysis syndrome,
active infection (sepsis) or presence of
disseminated intravascular coagulation

39. • He was not exposed to any other therapeutic
agents except CFZ, pomalidomide and
dexamethasone two months before
presentation and was in the middle of his
second cycle.
• Spontaneous recovery after discontinuation of
CFZ and thus his presentation was unlikely to
be TA-TMA.

40. Proposed Pathogenesis of
Proteasome Inhibitor Associated
Thrombotic Microangiopathy

41. Role of VEGF in TMA
• VEGF inhibition has been proposed as one of the
potential pathological factors responsible for
TMA associated with AKI
• VEGF is critical in maintenance and growth of
normal vasculature
• It is produced by podocytes in high concentration
and is considered to diffuse across the GBM
against the urinary flow and with its
concentration gradient to bind to its receptors on
glomerular endothelial cells
Eremina, V., et al., VEGF inhibition and renal thrombotic microangiopathy. N Engl J Med, 2008. 358(11): p. 1129-
36
Eremina, V. and S.E. Quaggin, Biology of anti-angiogenic therapy-induced thrombotic microangiopathy. Semin
Nephrol, 2010. 30(6): p. 582-90

42. Role of VEGF in TMA
• Podocytes and
glomerular endothelial
cells use VEGF-VEGF-R2
signaling for
transmembrane
communication, which
is essential for
structural and
functional integrity of
glomerular
endothelium and
fenestrations
Clinicopathological spectrum of kidney diseases in cancer patients treated
with vascular
endothelial growth factor inhibitors: a report of 5 cases and review of
literature
Joichi Usui MD, PhDa,b, Ilya G. Glezerman MDc, Steven P. Salvatore MDa,
Chandra B. Chandran MDd, Carlos D. Flombaum MDc, Surya V. Seshan
MDa,⁎

43. Role of VEGF in TMA
• Any disruption of this
mechanism,
– production (Proteasome
inhibitors),
– bevacizumab (anti-VEGF
antibodies),
– pazopanib (inhibits VEGF
1,2 and 3) and
– sunitinib (VEGF tyrosine
kinase inhibitor)
can potentially lead to
kidney damage specifically
TMA and podocytopathy
causing proteinuria and
hypertension N Engl J Med 2008;358:1129-36.

44. Proteasome inhibitors effect VEGF
pathways via NF-κB pathways

45. NF-κB
• NF-κB (nuclear factor kappa-light-chain-enhancer
of activated B cells) is a protein involved in the
regulation of genetic transcription
• NF-κB activation is in response to various
extracellular signals
– reactive oxygen species,
– TNFα
– IL-1β,
– bacterial polysaccharides
– radiation

48. • IκB kinase (IKK) when activated by extracellular
signals described above, cause phosphorylation
of IκB leading to ubiquitination of IκB
• After IκB degradation, NF-κB is free to enter the
nucleus and influence DNA transcription
• Increase in activity of NF-κB promotes VEGF
expression
Oscillations in NF-kappaB signaling control the dynamics of gene expression.
Nelson DE1, Ihekwaba AE, Elliott M, Johnson JR, Gibney CA, Foreman BE, Nelson G, See V, Horton CA, Spiller
DG, Edwards SW, McDowell HP, Unitt JF, Sullivan E, Grimley R, Benson N, Broomhead D, Kell DB, White MR.
Science. 2004 Oct 22;306(5696):704-8.
NF-kappaB promotes iNOS and VEGF expression in salivary gland adenoid cystic carcinoma cells and enhances
endothelial cell motility in vitro. Zhang J1, Peng B Cell Prolif. 2009 Apr;42(2):150-61. doi: 10.1111/j.1365-
2184.2009.00588.x.

49. • Thus, proteasome inhibitors decrease NF-κB
levels in nucleus leading to decreased VEGF
production potentially predisposing to TMA
Kiriakidis, S., et al., VEGF expression in human macrophages is NF-kappaB-dependent: studies using
adenoviruses expressing the endogenous NF-kappaB inhibitor IkappaBalpha and a kinase-defective form of the
IkappaB kinase 2. J Cell Sci, 2003. 116(Pt 4): p. 665-74.
Shibata, A., et al., Inhibition of NF-kappaB activity decreases the VEGF mRNA expression in MDA-MB-231 breast
cancer cells. Breast Cancer Res Treat, 2002. 73(3): p. 237-43.
Greenberger, S., et al., Targeting NF-kappaB in infantile hemangioma-derived stem cells reduces VEGF-A
expression. Angiogenesis, 2010. 13(4): p. 327-35.

50. • Acknowledgements:
• Danielle G Holanda, MD
• Department of Pathology, University of Iowa
Hospitals and clinics.

51. Thank you