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Renal Pathology
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Case 1 -
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Thrombotic Microangyopathy - Anti-VEGF Therapy Induced
Laura Barisoni
Department of Pathology
New York University
NY
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Clinical History
31 year old African-American with renal disease of unknown etiology (possibly related to malaria
and/or hypertension).
December 2006:
He received a cadaveric kidney transplant on
December 3rd, 2006. A time zero biopsy revealed mild mesangial expansion suggestive of
possible early diabetic nephropathy – donor transmitted, and arteriolar hyalinosis. There was slow graft
function, and he was treated initially with thymoglobulin and then a regimen of Prograf, Cellcept, and
Steroids. His creatinine plateaued at about 1.8-2.0 mg/dl.
July 2007:
In July 2007, he presented with painless jaundice and
workup revealed multiple masses in the head of the pancreas consistent with pancreatic cancer.
August 2007:
He underwent a Whipple procedure in August of 2007.
His recovery was uncomplicated. Following this procedure, he was discharged home with a creatinine of
1.4 and no edema or proteinuria. His immune regimen was reduced to low-dose tacrolimus (blood trough
levels were in the 4-6 ng/mL range) and prednisone.
September 2007:
In early September he had a rise in his
creatinine which was treated with a bolus of steroids (500 mg) and his creatinine returned to 1.7-1.9
(prior to starting chemotherapy).
In late September 2007*, he started adjuvant chemotherapy on a regimen of Taxotere, Avastin, and
Gemzar given monthly.
October 2007:
On October 8th, his creatinine was 1.9 and he had 1+ edema.
November 2007:
Repeat labs on November 12th showed a rise in
creatinine to 2.7 and 3+ pedal edema. His protein to creatinine ratio at this time was 22.0 (1399 mg
protein/63.6 mg creatinine).
December 2007:
His edema continued
to worsen and his creatinine rose to 3.9 on December 6th. A renal biopsy was performed on December
13th to rule out rejection versus membranous glomerulopathy secondary to malignancy.
| Date | Prograf Levels | S. Creatinine | Edema & | Proteinuria |
| April 07: | 15.5 | 1.4 | - | - |
| May 07: | 18.4 | 1.4 | - | - |
| June 07: | 6.1 | 1.4 | - | - |
| July 07: | 6.1 | 1.4 | - | - |
| August 07: | 4.1 | 1.4 | - | - |
| Sept 07*: | 6.0 | 1.8 | - | - |
| Oct 07: | 4.1 | 1.9 | 1+ | 1+ |
| Nov 07: | 4.1 | 2.7 | 3+ | 3+ |
| Dec 07: | Renal biopsy | 3.9 | 3+ | 3+ |
Case 1 - Slide 1
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Renal Biopsy Findings
Sections stained with H&E, PAS, trichrome and silver showed two cores of cortex with a thick
capsule. There were approximately 25 glomeruli per level, two of which globally sclerotic. Some of the
remaining glomeruli were normal in size, others revealed extensive wrinkling and folding of the basement
membrane accompanied by marked hypertrophy and focal hyperplasia of overlying podocytes. On occasion
podocytes contained protein reabsorption droplets. Focal mesangiolysis was also noted. Other glomeruli
revealed extensive splitting of the glomerular basement membrane accompanied by foamy hypertrophic
endothelial cells occluding or sub-occluding the capillary lumina. In addition, focal thrombi were
present in glomerular capillary lumina and fragmented red blood cells within the glomerular tuft. One to
two glomeruli revealed nuclear debris within the glomerular tuft. The tubular interstitial compartment
is remarkable for extensive fibrosis with diffuse moderate and focally severe inflammation composed of
lymphocytes, monocytes, plasma cells, occasional neutrophils and rare eosinophils. Interstitial fibrosis
was accompanied by tubular atrophy and mild interstitial edema. Tubules were acutely injured and reveal
extensive flattening of the epithelium with regenerative changes with nuclear atypia, vacuolization
(large vacuoles) of the cytoplasm. Focal protein reabsorption droplets were also present. No
significant lymphocytic or neutrophilic tubulitis was identified. There were 10-11 arteries per level,
up to the arcuate size. Arteries revealed narrowing of the lumen, mucoid intima edema, and focal
duplication of the elastic lamina. Interlobular arteries revealed narrowing of the lumen with
sub-occlusion or occlusion. Fibrin thrombi with fragmented red blood cells within the arterial wall and
markedly swollen endothelial cells were present. A single interlobular artery reveals severe mucoid
intimal edema, fragmented RBC, fibrin, hypertrophy of endothelial cells and inflammation (endothelitis).
Arterioles also had thrombotic microangiopathic changes with fibrin thrombi and fragmented RBC.
Immunofluorescence performed on frozen sections of the cortex containing 2 glomeruli revealed 2+
linear staining in the glomerular basement membrane for IgG and 3+ segmental positive stain for IgM.
Albumin was 1+ linear in the tubular and glomerular basement membrane. Arteriolar intima was strongly
positive for IgM, C3, C1q, fibrinogen, and kappa and lambda light chains.
Immunostaining for C4D showed only 1+ to 2+ granular stain in approximately 20% of the parenchyma.
Strong positive stain was noted in endothelial cells of arteries and glomeruli.
Tissue processed for ultrastructural studies showed renal cortex containing a single glomerulus
approaching global sclerosis. On ultrastructural analysis the glomerular basement membranes were
extensively folded and wrinkled. Podocytes were acutely injured and revealed extensive foot process
effacement, accompanied by microvillous transformation. No electron dense deposits or tubulo-reticular
inclusions were identified. Endothelial cells are diffusely mildly swollen and had lost their normal
fenestration.
Differential Diagnosis :
The clinical history and the changes above described are consistent with severe thrombotic
microangiopathy (TMA) involving arteries and glomeruli. The differential diagnosis includes Prograf
toxicity, antibody-mediated rejection, and/or cellular rejection and, Avastin-induced endothelial damage,
malignant hypertension and HUS/TTP. The tubulo-interstitial changes also raise a question of BK
infection. However, BK infection generally does not present with nephrotic syndrome. Moreover, against
the hypothesis of BK virus infection is the negative immunohistochemistry. Prograf is known to cause
TMA, but the patient has been on low levels of Prograf for few months prior the onset of nephrotic
syndrome and increased serum creatinine. Antibody-mediated rejection is also in the differential
diagnosis, as it can manifest with proteinuria in addition to increase serum creatinine. But the
presence of only very focal granular staining for C4d is against this diagnosis; moreover the serology
for anti-donor antibodies (performed after the biopsy results) was negative. Given the timing of the
onset of proteinuria, edema and renal failure the thrombotic microangiopathic changes are most likely
secondary to the Avastin (anti-VEGF) therapy.
Because of the severe interstitial inflammation and the presence of very focal endothelitis in a
patient with low levels of Prograf, T-cell mediated vascular rejection was also considered in the
differential diagnosis or as a co-existing process. Typically T-cell mediated rejection does not present
with massive TMA or proteinuria, indicating that in this particular case two different processes were
occurring simultaneously. On the other hand it cannot be completely ruled out that the endothelitis is a
secondary phenomenon following primary endothelial cell injury.
Diagnosis
- Thrombotic microangyopathy – anti-VEGF therapy induced
- Endothelitis in a single interlobular artery and moderate interstitial inflammation suggestive of
T-cell mediated rejection
Discussion
Proteinuria is often present in transplant patients although not always detected on routine studies.
Proteinuria may reflect a recurrence of the original renal disease, most commonly focal segmental
glomerulosclerosis, collapsing glomerulopathy, membranous glomerulopathy or other glomerulonephritis, or
may indicate a de novo glomerular process with podocyte injury. Proteinuria may also reflect podocyte
damage occurring during an episode of antibody-mediated rejection, or secondary to hyaline arteriolopathy
or TMA. The later two conditions have been described in association with FK506 or cyclosporine toxicity.
TMA after kidney transplantation may occur as a recurrent disease in patients with previous hemolytic
uremic syndrome or may develop de novo. In most of the cases recurrent post-transplant TMA occurs early
after the transplant, whereas de novo TMA may occurs at any time. The incidence of thrombotic
microangyopathy varies between less the 1% of renal transplant recipients to 14%. Known causes of TMA in
renal allograft, in addition to drug toxicity, include ischemia-reperfusion injury, antibody mediated
rejection and viral infection [1]. In native kidney, TMA has also been described in association with
therapy with inhibitors of VEGF activity for treatment of colon, liver, pancreas, ovary, lung, breast and
renal cell carcinoma
[2,
3,
4,
5,
6,
7,
8].
Significant progress has been made in the last few years in cancer treatment by using inhibitors of
angiogenesis such as agents targeting the vascular endothelial growth factor (VEGF) signaling pathway.
Three drugs have been developed and approved by FDA for treatment of carcinoma arising from several
organs:
1) bevacizumab (Avastin), a humanized monoclonal antibody that selectively
blocks VEGF ligands;
2) sunitib malate (Sutent, SU11248), blocks VEGF receptors as well as inhibit
platelet-derived growth factor receptors and other receptor tyrosine kinases implicated in tumor growth
and metastatic progression;
3) sorafenib (Nexavar, BAY 43-9006), which also blocks VEGF receptors,
platelet-derived growth factor receptors and tumor cell proliferation.
VEGF has multiple functions including promoting endothelial cell proliferation, differentiation and
survival, mediates endothelium-dependent vasodilatation, induces microvascular hyper-permeability and
participates in extracellular matrix remodeling processes. VEGF (also called VEGF-A) belongs to a family
of multipotent cytokines together with VEGF-B, VEGF-C, VEGF-D, VEGF-E and placenta growth factor. VEGF
and its receptors are widely expressed and lack of normal function or expression of VEGF produces
numerous side effects such as hypertension, upper respiratory infection, stomatitis, exfoliative
dermatitis, gastro-intestinal toxicity, hypothyroidism, proteinuria, coagulation disorders and neurotoxic
effects [9].
During development VEGF is expressed in many cell types and highly expressed in podocytes. Podocytes
continue to express VEGF in adult life, although the absolute levels are decreased. Endothelial cells
are the target cells for VEGF and express two tyrosine kinases receptors for VEGF (VEGFR-1 and VEGFR-2).
The relationship between VEGF abnormal expression and glomerular damage has been investigated in vitro
and in vivo, with both animal models and in human biopsies. Numerous animal studies demonstrated that
VEGF signaling is necessary for the glomerular filtration barrier formation. In fact, in a murine model
of deletion of both VEGF genes selectively in podocytes, perinatal death and renal failure occurs as a
consequence of endothelial defect in migration, survival and proliferation. Loss of a single VEGF allele
in podocytes leads to endotheliosis, (the glomerular lesion seen in pre-eclampsia) proteinuria, renal
failure and glomerulosclerosis. Deletion of one or both VEGF genes also leads to disappearance of
endothelial cells in mature glomeruli and mesangiolysis [10]. Similarly, neutralization of circulating
VEGF by anti VEGF antibodies and soluble VEGFR-1 induces endothelial cell injury (loss of fenestration
and detachment of endothelial cells) as well as podocyte injury (reduced expression of nephrin and
altered slit diaphragm structure)
and proteinuria in mice [11]. Whereas it is intuitive that
neutralization of circulating VEGF results in endothelial cell damage, the mechanism of podocyte damage
resulting from decreased circulating VEGF is less clear. Podocytes in vitro express few VEGF receptors
including VEGFR-1, VEGFR-3, neuropilin-1 and neuropilin-2, but there are conflicting reports regarding
the presence of VEGFR-2 in podocytes, the critical receptor by which VEGF signaling occurs, suggesting
that podocyte injury is indirect and derived from loss or damage of endothelial cell [10]. Eremina and
colleagues described 6 patients treated with VEGF inhibitors who developed TMA and proteinuria. She also
demonstrated that time-specific podocyte deletion of VEGF expression resulted in proteinuria and TMA in
mice. At the onset of proteinuria podocytes appeared relatively well preserved, but as the disease
progressed, features of collapsing glomerulopathy (podocyte hypertrophy and hyperplasia and collapsed
capillary loops) appeared together with intracapillary thrombi, fragmented red blood cells, swollen
endothelial cells (TMA)
[2]. These data suggest that altered VEGF function by pharmacological or genetic
deletion results in TMA and collapsing features with both endothelial and podocyte damage, which
clinically is reflected by hypertension, renal failure and proteinuria. In this murine model of
time-specific VEGF deletion from podocytes as well as in the case here presented, it is intuitive that
endothelial cell injury occurs as a direct consequence of reduced circulating VEGF, but it is still
unclear why podocyte re-enter the cell cycle and proliferate and features of collapsing glomerulopathy
appear in the glomeruli. Whereas in other cases described in the literature of anti-VEGF-induced renal
damage the concomitant collapsing glomerulopathy may have been attributed to simultaneous use of
pamidronate [6], this is not the case in this murine model or in our patient, suggesting that the
podocyte proliferation and collapsing features may be secondary to ischemic processes [12] and perhaps
mediated by mitochondrial pro-proliferative activity [13].
In conclusions, several reports in the literature link therapy with inhibitor of VEGF activity with
TMA and severe podocyte injury in the kidney.
Whether the renal manifestation of TMA occur in native or transplanted kidney in patients treated with
anti VEGF agents, close look at the time of onset of the symptoms and other relevant clinical features
should guide the interpretation of the renal morphologic findings.
References
- Ponticelli, C. and G. Banfi, Thrombotic microangiopathy after kidney transplantation. Transpl Int, 2006. 19(10): p. 789-94.
- Eremina, V., et al., VEGF inhibition and renal thrombotic microangiopathy. N Engl J Med, 2008. 358(11): p. 1129-36.
- Frangie, C., et al., Renal thrombotic microangiopathy caused by anti-VEGF-antibody treatment for metastatic renal-cell carcinoma. Lancet Oncol, 2007. 8(2): p. 177-8.
- George, B.A., X.J. Zhou, and R. Toto, Nephrotic syndrome after bevacizumab: case report and literature review. Am J Kidney Dis, 2007. 49(2): p. e23-9.
- Izzedine, H., et al., Thrombotic microangiopathy and anti-VEGF agents. Nephrol Dial Transplant, 2007. 22(5): p. 1481-2.
- Miller, K.D., et al., Randomized phase III trial of capecitabine compared with bevacizumab plus capecitabine in patients with previously treated metastatic breast cancer. J Clin Oncol, 2005. 23(4): p. 792-9.
- Roncone, D., et al., Proteinuria in a patient receiving anti-VEGF therapy for metastatic renal cell carcinoma. Nat Clin Pract Nephrol, 2007. 3(5): p. 287-93.
- Stokes, M.B., M.C. Erazo, and V.D. D'Agati, Glomerular disease related to anti-VEGF therapy. Kidney Int, 2008. 74(11): p. 1487-91.
- Kamba, T. and D.M. McDonald, Mechanisms of adverse effects of anti-VEGF therapy for cancer. Br J Cancer, 2007. 96(12): p. 1788-95.
- Eremina, V., H.J. Baelde, and S.E. Quaggin, Role of the VEGF--a signaling pathway in the glomerulus: evidence for crosstalk between components of the glomerular filtration barrier. Nephron Physiol, 2007. 106(2): p. p32-7.
- Sugimoto, H., et al., Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria. J Biol Chem, 2003. 278(15): p. 12605-8.
- Stokes, M.B., C.L. Davis, and C.E. Alpers, Collapsing glomerulopathy in renal allografts: a morphological pattern with diverse clinicopathologic associations. Am J Kidney Dis, 1999. 33(4): p. 658-66.
- Barisoni, L., et al., Collapsing glomerulopathy associated with inherited mitochondrial injury. Kidney Int, 2008. 74(2): p. 237-43.
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