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Keywords:

  • ADAMTS13 activity;
  • ADAMTS inhibitor;
  • humoral rejection;
  • thrombotic microangiopathy;
  • von Willebrand factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Case Report
  6. Discussion
  7. References

A 9-year-old renal transplant recipient presented with elevated serum creatinine levels 4 years post-transplant renal biopsy revealed humoral rejection including lesions suggestive for thrombotic microangiopathy (TMA). He received methylprednisolone pulses followed by a normalization of serum creatinine. Two more steroid responsive acute rejection episodes occurred. Two months later he presented rapidly progressive life threatening symptoms including bilateral pyramidal syndrome and hemoptysis. Serum haptoglobin became undetectable at this time and platelet count decreased (70 000/μl), suggesting TMA. Cerebral MRI revealed generalized ischemic white matter lesions. ADAMTS13 activity decreased to <5%. Daily plasma exchanges (PE) resulted in immediate improvement. All attempts to discontinue PE were unsuccessful. Transplantectomy resulted in normalization of generalized symptoms, hemolysis and ADAMTS13 activity (110%).

Multi-organ involvement has never been reported in acquired ADAMTS13 deficiency post-transplant. Rapid resolution after transplantectomy might suggest that renal TMA was responsible for acquired ADAMTS13 deficiency and thereby triggered the generalization of TMA lesions.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Case Report
  6. Discussion
  7. References

Thrombotic microangiopathy (TMA) is a well-described complication of bone marrow (1) and solid organ transplantation (2,3). In most of the reported cases, TMA post-transplant was associated with anti-calcineurin medication. Vascular rejection (4) and cytomegalovirus infection (5) have been reported as initiating factors. Endothelial cell injury is the crucial event in TMA leading to organ failure, microangiopathic hemolytic anemia and thrombocytopenia, which is assumed to be a consequence of platelet consumption at sites of endothelial injury.

Congenital abnormalities in complement system components such as factor H (6,7), I (8), or CD46 (9) lead to activation of the alternative pathway and has been shown to produce TMA, with a potential relapse post-transplant. Congenital abnormalities in vitamin B12 metabolism also known as cobalamin C disease are also known to provoke TMA (10,11) but have not been reported post-transplant.

Severe functional deficiency of the specific von Willebrand factor-cleaving metalloprotease (termed ADAMT13) activity has been shown to cause TMA, and more specifically thrombotic thrombocytopenic purpura (TTP) (12–14). Both inherited (15,16) and acquired ADAMTS13 (17) deficiencies have been described. Acquired forms are usually related to immune phenomena resulting in circulating auto-antibodies (Abs) directed either against ADAMTS13 itself or, presumably, against proteins essential for proper function of ADAMTS13 (17–20). Anti-ADAMTS13 auto-Abs can be detected in vitro either functionally because of their inhibitory effect on ADAMTS13 enzymatic activity (21,22) or, more recently, physically as immunoglobulin (Ig) G or IgM by enzyme-linked immunosorbent assay (ELISA) (18,23). In more than 80% of acquired TTP, anti-ADAMTS13 Abs are inhibitory IgG (14,21). In some cases, the mechanisms for acquired TTP may be different involving either anti-ADAMTS13 non inhibitory IgG or IgM (18,23) or circulating inhibitors not related to an IgG or an IgM (24).

Multi-organ involvement is a potential risk in all above-mentioned pathologies. However, in most of the reported cases lesions are limited or predominant in one organ.

Reversible acquired ADAMTS 13 deficiency has been reported once after renal transplantation (25). However, a generalized TMA post renal transplant due to acquired deficiency of ADAMTS13 activity has not yet been reported.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Case Report
  6. Discussion
  7. References

Biochemical investigation

ADAMTS13 activity was measured in serum as previously described (26). Briefly, the method consists in the hydrolysis of a constant amount of recombinant wild-type von Willebrand factor used as substrate by serial dilutions of tested patient serum used as ADAMTS13 provider. After hydrolysis, residual recombinant wild-type von Willebrand factor antigen is estimated using a two-site ELISA using monoclonal antibodies specifically directed to von Willebrand factor N-terminal part and C-terminal part, respectively.

Circulating inhibitor against ADAMTS13 activity was assayed as previously described (26) by measuring the residual ADAMTS13 activity in mixtures of patient serum and normal serum at three distinct volume: volume ratios, 1:1, 2:1 and 3:1, after a 30-minute pre-incubation at room temperature. Titer was semi-quantitatively defined as high, medium or low for residual ADAMTS13 activity lower than 5% in 1:1, 2:1 and 3:1 mixtures, respectively.

Anti-ADAMTS13 IgG in serum were detected using the commercial kit TECHNOZYM ADAMTS-13 INH ELISA (Technoclone GmbH, Vienna, Austria) using recombinant wild-type ADAMTS13 for coating and Abs to human IgG for staining (normal range < 12 units/mL).

ADAMTS13 antigen in serum was measured with the commercial kit IMIBUND® ADAMTS13 ELISA (American Diagnostica, Inc., Stamford, CT using a rabbit polyclonal antibody against ADAMTS13 (normal range 350–730 ng/mL).

Serum samples were investigated for anti-endothelial cell antibodies using an enzyme linked immunosorbent assay on endothelial/epithelial hybridoma cell line EAHy 926 (27) according to the technique described by Heurkens et al. (28).

Complement components and regulators were investigated as previously reported (29)

Case Report

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Case Report
  6. Discussion
  7. References

A male neonate was admitted with nephrotic syndrome related to Denys-Drash syndrome with diffuse mesangial sclerosis. He reached end-stage renal failure at the age of 15 months requiring hemodialysis and received a cadaveric kidney transplant at the age of 5 years with a standard immunosuppressive protocol including basiliximab induction, cyclosporine A (CyA), mycophenolate mofetil (MMF) and prednisone. Follow-up was uneventful until the age of 9 years, serum creatinine was 65 μmol/L, proteinuria was negative and blood pressure normal. No infectious or thrombembolic complications were noted.

At the age of 9 years the patient was admitted for sudden bilateral blindness, and elevated serum creatinine level. No signs of infectious retinitis were observed. Non-compliance to immunosuppressive treatment was strongly suspected as several cyclosporine trough levels were unusually low and the family background became unstable. The kidney biopsy (Figure 1) revealed humoral vascular rejection (c4d positive) including arteriolar and capillary thrombi, glomerular capillary wall thickening and double contours suggesting TMA. He received three pulses of methyl-prednisolone (1 g/1.73 m2 on day 1, 3, 5) followed by normalization of serum creatinine on day 10. His immunosuppressive regime was CyA (5 mg/kg/day), MMF (600 mg/m2 twice daily) and prednisone (1 mg/kg/day for 3 weeks followed by progressive tapering down to 0.5 mg/kg/day). The CMV antigen, quantitative Epstein-Barr virus (EBV) and BK virus polymerase chain reaction (PCR) were negative and remained negative throughout the entire follow-up period. Two more acute rejection episodes occurred, all were steroid responsive, and baseline serum creatinine remained satisfactory (75 μmol/L). Two months later the boy presented with malaise, severe abdominal pain, vomiting and generalized abdominal defense, concomitant with elevated serum creatinine. A second kidney biopsy revealed similar lesions as the first one (humoral vascular rejection and thrombotic microangiopathy). He received three methylprednisolone pulses and intravenous immunoglobulins. His general condition remained unstable and 2 weeks later he presented rapidly progressive symptoms including severe asthenia, CNS involvement with bilateral pyramidal syndrome, dyspnoea, hemoptysis and severe abdominal pain. At this time serum haptoglobin levels became undetectable, serum creatinine was 200 μmol/L and platelet count was 70 000/μL, suggesting TMA to be responsible for the generalized symptoms.

image

Figure 1. Time course of clinical and biological parameters. MP = methyl-prednisolone pulse (1000 mg/m2 on day 1, 3, 5); IV-Ig = intravenous immunoglobulins; RB = renal biopsy.

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Surprisingly, ADAMTS13 activity in serum was <5% concomitant with a circulating inhibitor (medium titer) not related, however, to an anti-ADAMTS13 IgG (undetectable titer); further, ADAMTS13 antigen level in serum was found in the normal range at 600 ng/mL. In contrast, factor H, I, B and CD46 were normal. No anti-HLA antibodies and no anti-endothelial cell antibodies were detected. Cerebral MRI revealed generalized ischemic white matter lesions presumably related to cerebral TMA. Daily plasma exchanges (PE) were started with 1.5 plasma volume exchanged per session, resulting in immediate improvement of all symptoms. From day 1 to 4 plasma was exchanged against fresh frozen plasma (FFP), from day 5 against albumin 4% and every 4 days against FFP. PE were performed on a daily basis until day 7. We tapered down the frequency of PE, resulting in reappearance of all previously described symptoms within 48 h after the last plasma exchange. There was no significant difference in efficacy between PE against albumin 4% or FFP. Discontinuation of cyclosporine over 1 week did not result in clinical or biological improvement. A second attempt to discontinue PE was unsuccessful and transplantectomy was performed after a third kidney biopsy confirming lesions of TMA similar to the first two biopsies. All neurological, abdominal and pulmonary symptoms disappeared completely after nephrectomy and no further PE was necessary. A summary of all laboratory investigations is given in Table 1.

Six months after transplantectomy clinical examination and laboratory analyses are normal without evidence for persisting TMA. The patient is currently on peritoneal dialysis awaiting re-transplantation.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Case Report
  6. Discussion
  7. References

TMA, a multi-system disorder characterized by thrombocytopenia and hemolytic anemia results in ischemic organ dysfunction. Antibody-mediated acquired reduction of ADAMTS13 activity has been found in cases of TMA clinically presenting generally as thrombotic thrombocytopenic purpura (TTP) (19–21,30,31). These cases of TMA were either idiopathic or secondary to infection, autoimmune disease, pregnancy, or drugs. Post-transplant TMA is usually not related to acquired deficiency of ADAMTS13 activity, but rather to calcineurine inhibitor toxicity (32,33). A severe acquired deficiency of ADAMTS13 activity has been reported in one case of TMA after kidney transplantation (25). In this patient an ADAMTS13 inhibitor (IgG against ADAMTS13) was found. Plasma exchanges and discontinuation of cyclosporine resulted in clinical improvement and the ADAMTS 13 inhibitor disappeared. However, this patient did not present multi-organ involvement and no rejection episode was suspected as an initiating factor.

Calcineurine inhibitors are suspected to increase autoimmune phenomena in particular the production of auto-reactive T cells that can induce vascular damage in the transplant (34). It is not clear if autoimmune phenomena related directly or indirectly with ADAMTS13 activity contribute to the pathogenesis of calcineurine inhibitor related TMA post-transplant.

It can be hypothesized that vascular rejection stimulates immunization against endothelial antigens as antigen exposure is increased. Figure 3 shows a hypothetic model for the interaction of humoral rejection and decrease of ADAMTS13 activity by immunization against components of the ADAMTS13 system. Interestingly, ADAMTS13 has recently been shown to be synthesized and secreted from endothelial cells (35). In that regard, our patient's serum contained an ADAMTS13 circulating inhibitor of unknown origin as this latter was not identified as an IgG. In addition, although ADAMTS13 activity was undetectable, ADAMTS13 antigen was normal. Thus, several hypothesis can be debated to explain the ADAMTS13 inhibitory activity detected in our patient's serum. The first and most likely hypothesis is the presence of another anti-ADAMT13 Ig isotype (i.e. IgM (18) or IgA) that would specifically inhibit ADAMTS13 catalytic site without involving an immune-depletion mechanism. The second hypothesis is the development of an Ab targeting not ADAMTS13 itself but another protein crucial for ADAMTS13 activity in serum.

image

Figure 3. Hypothetic model of interaction between vascular rejection and immunization against ADAMTS13. Vascular rejection results in endothelial damage and may stimulate immune phenomena against the ADAMTS13 system. The secondary decrease of ADAMTS13 activity might contribute to further endothelial damage resulting in a vicious circle. Transplantectomy results in rapid clinical and biological remission suggesting the crucial role of the renal allograft as a trigger for generalized microangiopathy.

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Indeed, in our patient, vascular rejection is likely to have triggered auto(allo)-Abs production against specific proteins present in the micro-environment of endothelial cells and thus potentially involved in ADAMTS13 regulation in vivo.

In our case, however, several mechanisms already described or suggested in the literature appear very unlikely considering the normal ADAMTS13 antigen level in our patient's serum: first, an in vitro inhibition of ADAMTS13 activity by hemoglobin (24) can be excluded as the serum sample was not hemolyzed. Second, the development of an auto-Ab against a candidate protein for an ADAMTS13 endothelial receptor (i.e. CD36) is unlikely as this mechanism would be associated with an increased degradation of the circulating soluble form of ADAMTS13 leading to a decreased ADAMTS13 antigen serum level. Third, a degradation of ADAMTS13 by enzymes like plasmin, thrombin (36) or granulocyte elastase (37) would also be associated with decreased ADAMTS13 antigen levels in serum.

Uncontrolled humoral vascular rejection and acquired ADAMTS13 deficiency in our patient seems to be closely related. Therefore, it can be speculated, that acquired ADAMTS13 deficiency might be under-diagnosed in the setting of vascular rejection and might be a trigger enhancing endothelial damage in the allograft.

Multi-organ involvement in a vascular rejection context has never been reported in acquired ADAMTS13 deficiency. The rapid regression of all systemic TMA associated symptoms including cerebral TMA after transplantectomy suggests that renal TMA played a key role in the maintenance of generalized TMA. The initial TMA lesion was located in the kidney transplant responsible for severe organ failure. Furthermore, the abundance of micro-vessels in the kidney suggests that from a quantitative point of view, the kidney transplant can be considered as the predominant site of endothelial damage. This may result in either further consumption of fibrinolytic proteins or as a trigger of auto-immune processes responsible for the production of inhibitory antibodies directed against the ADAMTS13 system.

The mechanism of endothelial damage presumably depends on more than only auto-antibodies, as the time interval between transplantectomy and regression of generalized TMA was shorter (about 3 days) than antibody half life time.

Other contributing factors, directly derived from the renal endothelium might play a role. However, as three plasma exchanges were performed directly prior to nephrectomy it can also be presumed that only low levels of inhibitory antibodies were present at the time of nephrectomy and once the transplant removed there was no further antibody production.

In acquired TMA, plasma exchanges may increase plasma ADAMTS13 activity either by providing exogeneous ADAMTS13 or by clearing inhibitory antibodies and other degradation products. No difference has been noted between plasma exchange against albumin 4% or against fresh frozen plasma, suggesting the role of PE to be rather increased removal of a potential inhibitor rather than supplementation.

Treatment with anti-CD 20 antibodies might be an interesting option, potentially reducing immune phenomena in vascular rejection. Production of ADAMTS13 inhibitory antibodies may be reduced, but no rapid clearance of existing antibodies can be expected. The rapidly increasing severity of generalized TMA in our patient required rapid efficacy against TMA and did not allow us to use this treatment option.

The possibility of re-transplantation has been discussed for our patient. Pre-existing ADAMT13 inhibitory antibodies might increase the risk of endothelial damage in a new renal allograft. Further, calcineurine inhibitor doses are comparatively high in the first months post-transplant. The use of calcineurin inhibitors is potentially dangerous and should be very carefully monitored in this setting. However, a time interval of approximately 1 year between transplantectomy and re-transplantation (considering the normalization of serum ADAMTS13 activity as well as all indirect parameters of TMA), suggests re-transplantation to be a treatment option with a reasonable benefit-risk ratio.

In conclusion, this is the first report of generalized thrombotic microangiopathy attributed to acquired deficiency of ADAMTS13 activity post-transplant. Investigation of the ADAMTS13 system in transplant recipients with vascular rejection episodes might add to the understanding of endothelial damage in these settings.

References

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  2. Abstract
  3. Introduction
  4. Methods
  5. Case Report
  6. Discussion
  7. References
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