De novo thrombotic microangiopathy after kidney transplantation

    De Novo Thrombotic Microangiopathy after Kidney Transplantation Neetika Garg, Helmut G. Rennke, Martha Pavlakis, Kambiz Zandi-Nejad PII: DOI: Reference:

S0955-470X(17)30005-8 doi: 10.1016/j.trre.2017.10.001 YTRRE 462

To appear in:

Transplantation Reviews

Please cite this article as: Garg Neetika, Rennke Helmut G., Pavlakis Martha, ZandiNejad Kambiz, De Novo Thrombotic Microangiopathy after Kidney Transplantation, Transplantation Reviews (2017), doi: 10.1016/j.trre.2017.10.001

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ACCEPTED MANUSCRIPT 1 Title Page

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De Novo Thrombotic Microangiopathy after Kidney Transplantation

Department of Medicine, Nephrology Division, Beth Israel Deaconess Medical Center/ Harvard

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Neetika Garg MDA,1, Helmut G. Rennke MDB, Martha Pavlakis MDA, Kambiz Zandi-Nejad MDA

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Medical School, Boston, MA 02215

Department of Pathology, Brigham and Women’s Hospital/ Harvard Medical School, Boston,

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Present Address: Department of Medicine, Nephrology Division, University of Wisconsin School

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Neetika Garg MD

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Corresponding author:

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of Medicine and Public Health, Madison, WI 53705

Email address: [email protected]

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University of Wisconsin School of Medicine and Public Health 1685 Highland Ave

4171 Medical Foundation Centennial Building Madison, WI 53705 Phone number: +1 6176507026

Running title: De novo TMA after kidney transplantation

ACCEPTED MANUSCRIPT 2 De Novo Thrombotic Microangiopathy after Kidney Transplantation Abstract

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Thrombotic microangiopathy (TMA) is a serious complication of transplantation that adversely

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affects kidney transplant recipient and allograft survival. Post-transplant TMA is usually classified into two categories: 1) recurrent TMA and 2) de novo TMA. Atypical hemolytic uremic

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syndrome (aHUS) resulting from dysregulation and over-activation of the alternate complement

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pathway is a rare disease but the most common diagnosis associated with recurrence in the allografts. De novo TMA, on the other hand, represents an overwhelming majority of the cases

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of post-transplant TMA and is a substantially more heterogeneous entity than recurrent aHUS. Here, we review the etio-pathogenesis, diagnosis and treatment options for de novo post-

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transplant TMA. It is usually in the setting of calcineurin inhibitor use, mammalian target of rapamycin inhibitor use, or antibody mediated rejection; recently genetic mutations in

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complement regulatory genes for Factor H and Factor I similar to those described in aHUS have

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been reported in up to a third of these patients. Systemic signs of TMA are frequently absent, and a renal allograft biopsy is often needed to establish the diagnosis. Although withdrawal of

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the offending agents is usually the first line of treatment and resolution of laboratory abnormalities has been documented with this approach in several case reports and case series, available retrospective data demonstrate lack of benefit in long-term graft outcomes. Costimulation blockage with belatacept provides an effective alternate immunosuppressive strategy for these patients. Anti-complement therapy with eculizumab is effective in some cases; further work is required to define which patients with TMA (with and without concomitant antibody-mediated rejection) would benefit from receiving this treatment, and what biomarkers can be used to identify them.

ACCEPTED MANUSCRIPT 3 Keywords Thrombotic microangiopathy; hemolytic uremic syndrome; kidney transplantation; belatacept;

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eculizumab; complement

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Abbreviations aHUS: Atypical hemolytic uremic syndrome

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AMR: Antibody mediated rejection

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CKD: Chronic kidney disease CNI: Calcineurin inhibitor

GFR: Glomerular Filtration Rate

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HUS: Hemolytic uremic syndrome

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ESRD: End stage renal disease

IVIG: Intravenous immunoglobulin

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LDH: Lactate dehydrogenase

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MCP: Membrane cofactor protein

mTOR: Mammalian target of rapamycin

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PG: Prostaglandin

PLEX: Plasma exchange PTC: Peri-tubular capillary TMA: Thrombotic microangiopathy TTP: Thrombotic thrombocytopenic purpura USRDS: United States Renal Data System VEGF: Vascular endothelial growth factor Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

ACCEPTED MANUSCRIPT 4 1. Introduction

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Thrombotic microangiopathy (TMA) is a well-recognized and serious complication of

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transplantation that is associated with poor graft and patient outcomes. A United States Renal Data System (USRDS)-based study of 15,870 renal transplant recipients, the largest study

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evaluating the epidemiology and outcomes in post-transplant TMA to date, reported a total

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incidence density of 5.6 episodes per 1,000 persons-years. Although graft survival was not analyzed, patient mortality was reported at approximately 50 percent at three years after the

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diagnosis was established.[1] While therapeutic plasma exchange was available at the time patients in this study were transplanted, the USRDS database does not reliably capture whether

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this treatment option was utilized and hence, its impact on outcomes could not be analyzed.

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Post-transplant TMA is usually classified into two categories: 1) recurrent disease, where the same disease process that manifests as TMA in the native kidney re-develops in the allograft;

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and 2) de novo TMA after transplantation, where TMA develops for the first time in patients

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who have never had any evidence of the disease prior to transplantation [Table 1]. It is worth emphasizing that the diagnosis of aHUS may be missed in the native kidneys in the absence of systemic signs of a microangiopathic process, and subsequently a recurrence in the allograft may be misclassified as de novo TMA. The availability of eculizumab for not only treatment but also prevention of aHUS recurrences in the renal allografts makes this distinction clinically extremely important. As such, diagnosis of TMA in the allograft must prompt re-evaluation of the underlying etiology if not already definitely established. Particular attention must be paid to the underlying etiology of end stage renal disease (ESRD) as TMA can complicate various glomerulonephritis and small vessel vasculitis syndromes.[2]

ACCEPTED MANUSCRIPT 5 The risk of recurrence of TMA in the allograft depends on the etiology of primary TMA affecting the native kidneys. Atypical hemolytic uremic syndrome (aHUS), a rare disorder resulting from

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over-activation of the alternate complement pathway at cell surface is the most common

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diagnosis associated with recurrence. The precise risk of recurrence is determined by the specific underlying abnormality present in an individual case.[3] For example, factors H and I are

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circulating complement regulatory proteins manufactured predominantly in the liver. In patients

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with mutations in respective genes CFH and CFI, production of abnormal factors H and I persists after kidney transplantation resulting in a 70 to 90 percent recurrence rate.[3, 4] In contrast,

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membrane cofactor protein (MCP) is a trans-membrane complement regulatory protein that is synthesized locally in the renal endothelial cells. After transplantation, the donor endothelial

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cells in the transplanted organ are able to produce normal MCP and therefore, TMA recurrence in aHUS patients with MCP gene mutations depends on whether any additional complement

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regulatory defects are present.[3-5] In a study by Bresin et al., 22.6% of patients with MCP

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mutations carried additional mutations in other complement genes. One-third (4/12) of the allografts were lost to recurrence in presence of other concomitant pathogenic mutations, as

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opposed to only one of 13 with isolated MCP mutations. Overall, TMA recurs in the allograft in 60 percent of the patients with aHUS. If untreated, recurrent aHUS leads to graft loss in 90 percent of affected patients, 80 percent of these occur within the first year after transplantation.[6] On the other hand, typical hemolytic uremic syndrome (HUS) caused by infection with shiga-toxin producing bacteria is associated with a generally favorable renal prognosis. Among the few patients that progress to ESRD and subsequently undergo renal transplantation, allograft survival is excellent and there is no risk of TMA recurrence.[7-9] Thrombotic thrombocytopenic purpura (TTP) is the second major diagnosis under the classification of TMA. It results from genetic or acquired deficiency of a disintegrin and

ACCEPTED MANUSCRIPT 6 metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), a vonWillebrand factor-cleaving protease. Before serologic assessment of ADAMTS13 activity became

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easily available, the distinction between TTP and HUS relied mostly on whether neurologic or

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renal dysfunctions predominated; presence of moderate to severe renal insufficiency was considered adequate to establish a diagnosis of HUS. However, significant overlap exists

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between clinical manifestations of these two conditions. In a recent analysis of 92 patients

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diagnosed with TTP based on low ADAMTS13 activity, acute kidney injury was present in more than half the patients, of which 50 % developed chronic kidney disease (CKD) or ESRD.[10]

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Although very little is documented about the risk of recurrence in this population, it is plausible that TTP may recur in the allograft given the persistence of the underlying defect after

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transplantation.[11] Similarly, lupus nephritis (with or without documented anti-phospholipid antibodies) is associated with TMA in 5-10% of the cases and has been documented to recur in

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the allograft in case report and small case series and has been documented to recur in the

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allograft.[12-14]

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While ESRD due to aHUS, which is believed to be under-diagnosed, has been identified as the strongest risk factor for TMA recurrence, it should be noted that aHUS is still a rare disorder and de novo TMA constitutes the great majority of the reported cases of post-transplant TMA. In the USRDS-based study by Reynolds et al mentioned earlier, the risk of post-transplant TMA was 36.5 times higher among recipients with ESRD resulting from HUS as compared to other causes (29.2% vs 0.8%); however, the absolute number of patients with recurrent TMA was only 12 as compared to 112 for patients with de novo TMA.[1] In another analysis by Langer et al where TMA was diagnosed based on laboratory parameters, the incidence of de novo TMA was reported at 1.5%.[15] Furthermore, studies that include both clinical and histologic analyses for establishing the diagnosis of de novo TMA report a much higher incidence at 3 to 14%[Table

ACCEPTED MANUSCRIPT 7 2].[16, 17] These data suggest that de novo TMA is a more common complication of renal transplantation than usually appreciated. Allograft outcomes in affected patients are generally

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considered to be poor, with graft loss rates as high as 40% at 2 years.[17, 18] In this article, we

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review the literature on etio-pathogenesis of de novo TMA, clinical presentation and diagnostic

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criteria, and therapies evaluated for its management in affected patients.

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2. Etio-pathogenesis of de novo TMA after renal transplantation

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Although the pathogenesis of post-transplant de novo TMA in renal allografts is still poorly understood, drug toxicity related to calcineurin inhibitors (CNIs) and mammalian target of rapamycin (mTOR) inhibitors, and antibody-mediated rejection (AMR) are implicated in a vast

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majority of the cases; less common etiologies include infections and non-immunosuppressive

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drugs. Why only a small fraction of patients exposed to the same risk factors develop this complication is not fully understood, one study documents underlying complement regulatory

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abnormalities in a third of these cases.[19]

2.A. CNI-associated TMA: The association between CNIs, both cyclosporine and tacrolimus, and de novo TMA is well documented in the literature. When TMA first develops in the allograft in a renal transplant recipient on a CNI, it is assumed as a matter of course that the CNI is the culprit. However, several lines of evidence suggest that cyclosporine or tacrolimus exposure alone is not sufficient to cause this complication. More than 95 percent of renal transplant recipients receive cyclosporine or tacrolimus; only a small minority of them develops TMA suggesting presence of an underlying predisposing factor in these patients.[20] Temporary or permanent discontinuation of CNI after development of de novo TMA in the allograft has not been consistently shown to improve long term graft outcomes.[18] Furthermore, a USRDS-based

ACCEPTED MANUSCRIPT 8 study showed that the incidence of TMA is in fact significantly higher in patients who did not receive initial maintenance CNI treatment (11.9/1,000 patient-years) as compared to patients

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who were placed on initial maintenance CNI therapy (5.0/1,000 patient-years).[1] While

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potential confounding by indication limits interpretation of these data, this does emphasize that

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several factors other than CNI exposure are at play in pathogenesis of de novo TMA.

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Various mechanisms are believed to contribute to development of TMA with CNI use. First, the loss of normal equilibrium between vasoactive peptides (increased biosynthesis of

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vasoconstrictor substances such as thromboxane A2 and endothelin, and reduced expression of vasodilatory molecules such as prostaglandin (PG) E2 and prostacyclin (PGI2)) leads to arteriolar

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vasoconstriction.[21, 22] The resulting renal ischemia can cause endothelial injury.[23] Secondly, platelet-activating, pro-coagulant and anti-fibrinolytic effects of CNIs have been implicated in

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the development of TMA, especially when the endothelium is already injured as a result of other

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mechanisms such as ischemia-reperfusion injury, antibody mediated rejection, etc.[23-25] Thirdly, cyclosporine causes the endothelial cells to release microparticles that activate the

TMA.[26]

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alternate complement pathway, a mechanism already known to be involved in pathogenesis of

2.B. mTOR inhibitor-associated TMA: There is increasing evidence that sirolimus and everolimus are associated with de novo TMA in renal transplant recipients. Sirolimus forms a complex with FK-binding protein that binds with high affinity to mTOR. In addition to blocking cell cycle progression and proliferation, mTOR inhibition also leads to decreased renal expression of vascular endothelial growth factor (VEGF) and death of endothelial progenitor cells; these latter anti-angiogenic effects are believed to contribute to the pathogenesis of TMA.[27, 28] A recent study by Keir et al. demonstrated that VEGF inhibition leads to decreased Factor H synthesis in

ACCEPTED MANUSCRIPT 9 the kidney, and that podocytes carrying CFH genetic variants are less efficient at inhibiting the alternate complement pathway and therefore potentially more susceptible to reduction in

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Factor H levels in the presence of VEGF antagonism.[29] As discussed later in section 2.E, CFH

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mutations have been described in kidney transplant recipients with de novo TMA; these patient may be especially vulnerable to developing this complication in the face of m-TOR inhibitor

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use.[19] In addition, increased procoagulant and reduced fibrinolytic state associated with these

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medications may also play a role.[30, 31] The precise risk for post-transplant TMA attributable to mTOR inhibitors is unclear.[15, 32, 33] Some studies suggest that this risk may even be higher

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than with CNIs.[1, 34] However, confounding by indication (i.e. sirolimus may have been used as

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rescue therapy after diagnosis of TMA) limits interpretation of these data.[1, 34]

There are several studies demonstrating that the risk of developing TMA is higher with a

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combined CNI and mTOR inhibitor immunosuppressive regimen as compared to either

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medication alone. Fortin et al evaluated risk of TMA with four immunosuppressive regimens (cyclosporine + mycophenolate, cyclosporine + sirolimus, tacrolimus + mycophenolate,

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tacrolimus + sirolimus) in a single center study of 368 kidney or kidney-pancreas transplant recipients. The risk was the highest in the groups where a CNI and an m-TOR inhibitor were used together: incidence of 20.7% and relative risk of 16.1 in the cyclosporine + sirolimus group and incidence of 6.1% and relative risk of 6.1 in the tacrolimus + sirolimus group.[32] In another study of 396 kidney transplant recipients by Nava et al, 36(7.3%) patients developed TMA; 17 of these were drug-related. Interestingly, not only were the CNI and the m-TOR inhibitor levels higher in the TMA group compared to those who did not develop this complication, the sum of concentrations of these two drugs was also higher in the former group (cyclosporine + everolimus: 15.2±6.3 ng/dL in those with TMA vs 10.7±2.1 ng/dL in those without TMA, and

ACCEPTED MANUSCRIPT 10 tacrolimus + everolimus: 21.1±11.3 ng/dL in those with TMA vs 9.6±1.3 ng/dL in those without TMA. Endothelial damage by CNI, in setting of mTOR inhibitor related impediment to repair of

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endothelial injury is postulated as the reason for this additive risk.[32, 35, 36] Based on these

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studies, we recommend caution with use of combination of these two classes of medications in the early post-transplant period when higher therapeutic levels are desirable, or in the presence

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of other risk factors for endothelial injury, for example presence of mutations in complement

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regulatory genes.[19]

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2.C. AMR-associated TMA: AMR is a common and important cause of post-transplant TMA. Endothelial cells in the renal allograft are the primary target of recipient’s alloimmune response.

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Histopathologic characteristics of acute AMR range from endothelialitis to necrotizing vasculitis; systemic or renal-limited TMA can occur as well.[37, 38] In a study by Meehan, peritubular

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capillary (PTC) C4d staining (a surrogate marker of AMR) was observed in 6 (16.2%) of 37

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biopsies with TMA.[1, 39] Conversely, TMA was present in only 6 (3.3%) of 182 biopsies with PTC C4d positivity. In another study of 960 patients by Satoskar et al, more than half (33/59; 55%) of

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de novo TMA patients had diffuse PTC C4d staining.[18] While cyclosporine was used in a large majority of the patients in this study and may have had an additive effect in the development of TMA, the difference in the prevalence of TMA in C4d positive biopsies (33/245; 13.6%) vs TMA in C4d negative biopsies (26/715; 3.6%) suggests that humoral rejection itself plays a role in pathogenesis of post-transplant TMA. Furthermore, both these studies showed that concurrent immunopathologic findings of AMR and TMA portend a worse allograft outcome than AMR alone.[18, 40]

The distinction whether de novo TMA develops in the renal allograft over the background of

ACCEPTED MANUSCRIPT 11 AMR or by itself has critical therapeutic implications. TMA attributed to immunosuppressive drug toxicity is usually treated by decreasing the dose of culprit medication or changing to a

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different immunosuppressive regimen (even though data documenting long-term benefit with

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this intervention are lacking).[18] On the other hand, various other therapeutic options targeting the underlying pathophysiology are available for patients with AMR, with or without

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associated TMA.

2.D. Other causes: Uncommon etiologies associated with TMA that have been reported in

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literature include various viral infections such as chronic hepatitis C infection (with or without associated anti-cardiolipin seropositivity) [41, 42] cytomegalovirus [43, 44], parvovirus [45, 46]

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and BK virus [47]; antiviral therapy with ribavirin/interferon [48] and leflunomide [49]; and disseminated histoplasmosis. [50, 51]There are also rare case reports of acquired ADAMTS13

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deficiency presenting as de novo TMA after renal transplantation.[52, 53]

Ischemia-reperfusion injury is associated with complement activation and can amplify

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complement-mediated injury.[54] While frequently listed as a risk factor for post-transplant TMA, there are no studies that have systematically evaluated this as a risk factor. On the contrary, living donation has not found to be associated with protection against renal graft dysfunction.[17]

Additionally, phenotypical shifts between various disorders of alternate complement pathway dysregulation have been documented, where the mutation manifests as a C3 glomerulopathy in the native kidneys and as de novo aHUS after transplantation.[55, 56]

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2.E. Complement Regulatory Gene Abnormalities: All of the above mentioned risk factors are

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common in the transplant population. It remains unknown why only a relatively small

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percentage of the renal transplant recipients develop TMA, while an overwhelming large majority exposed to the same risk factors does not. One hypothesis is that only patients with an

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underlying susceptibility towards endothelial injury and/or pro-coagulant state develop this

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complication. While the precise pathways by which each of the previously discussed individual risk factors contribute to pathogenesis of TMA have not been fully delineated, Chua et al

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documented that complement activation in the kidney is the common denominator in this heterogeneous condition. They examined 42 renal sections with histologically confirmed

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diagnosis of TMA obtained from a heterogeneous group of 36 patients, and found C4d deposits in 88.1% of the samples and C4d deposits co-localizing with C5b-9 in 59.5%. Of note, the

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distribution of C4d deposits in TMA is different from the PTC C4d deposition characteristic of

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AMR. In TMA, these deposits were localized in the glomeruli in 76.2% of the samples, in the arterioles in 59.5% and in the PTCs in only 9.5%. In post-transplant TMA (12 of 42 specimens);

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C4d deposits were present in the glomeruli in 66.7%, in the arterioles in 83.3% and in the PTCs in only 25%.[57]

Le Quintrec et al. retrospectively recruited 24 kidney transplant recipients with post-transplant de novo TMA diagnosed based on presence of histo-pathologic evidence and at least one systemic manifestation (thrombocytopenia and/or microangiopathic hemolytic anemia with a negative Coomb’s test and/or acute renal failure). Six of 24 (25%) patients were found to have low C3 and/or low Factor B levels suggestive of alternate complement pathway activation. Additionally, a mutation in CFH or CFI gene was found in 7 of 24 (29%), two of whom had a

ACCEPTED MANUSCRIPT 13 mutation in both genes. No mutations were detected in 25 control kidney transplant recipients without TMA and in 100 control healthy individuals. [19] Factors H and I are the two major

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regulators of the alternate complement pathway. Factor H dissembles the C3 cleaving enzyme

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C3bBb as well as serves as a cofactor for Factor I, which inactivates C3b. Loss of function mutations in either or both of these genes can increase alternate complement pathway activity

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and cause endothelial injury. While patients with localized TMA in the renal allograft were not

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included in this study, these results suggest an overlap in pathogeneses of aHUS and de novo TMA, hitherto considered two separate clinical entities. Since this investigation was published in

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2008, mutations in several other genes involved in the complement and the coagulationfibrinolysis cascades have been identified in patients with aHUS.[58] However, to the best of our

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3. Clinical presentation

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knowledge, these have not been tested for in the post-transplant TMA population.

Post-transplant de novo TMA is most frequently diagnosed in the initial 3 to 6 months after

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transplantation; this propensity is often attributed to the high CNI trough levels targeted during this period.[1] However, it should be noted that the risk in the subsequent period still remains substantial and TMA can develop in a renal transplant recipient at any time.[17, 59]

The clinical presentation of post-transplant TMA is highly variable. Some patients develop the classic triad of the laboratory findings of systemic TMA: 1) micro-angiopathic hemolytic anemia (evidenced by drop in hemoglobin, elevated serum lactate dehydrogenase (LDH) enzyme level, low serum haptoglobin level and presence of schistocytes on peripheral smear), 2) absolute or relative thrombocytopenia, and 3) acute kidney injury. More often, only some of these

ACCEPTED MANUSCRIPT 14 abnormalities are present and in more than half the cases, TMA is limited to the allograft with slowly progressive renal insufficiency and/or worsening hypertension being the only clinical

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manifestations (frequently referred to as ‘localized TMA’).[16-18, 60] One study reported that

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localized TMA was diagnosed later in the post-transplant course than systemic TMA; however it is unclear whether this is because systemic TMA occurs early or because it is associated with

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more severe dysfunction leading to an earlier biopsy and diagnosis.[16] In patients with

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localized TMA, the diagnosis is frequently not apparent until an allograft biopsy is done[16],

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which remains the gold standard for establishing the diagnosis of post-transplant TMA.

The histopathological changes seen in patients with these disorders are rather nonspecific for

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the various clinical entities, and affect the glomerular and peritubular microcirculation and the arteries and arterioles. The combinations of the morphological changes allow us to classify the

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pathological process into acute (or active) and chronic thrombotic angiopathies. During the

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acute or active process there is dominance of endothelial cell injury, platelet aggregation and formation of thrombi, and to a minor extent active inflammation (Figure 2). In the chronic

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thrombotic angiopathies there is evidence of persistent endothelial injury and signs of remodeling of the glomerular capillary walls and the arterial and arteriolar intima and media; this process results in formation of duplicated basement membranes (double contours in the glomerular capillaries and multi-layering of the basement membrane of the peritubular capillaries) and increase in the layers of matrix and cells in the arterial and arteriolar walls that result in sclerosis often with characteristic onionskin lesions (Figure 3).

Lastly, diagnosis of post-transplant TMA should prompt re-evaluation of the etiology affecting the native kidneys in an individual patient. Similar to post-transplant TMA, some patients with

ACCEPTED MANUSCRIPT 15 aHUS do not show systemic signs of TMA and the underlying diagnosis may not be obvious. In the absence of a native kidney biopsy, these patients are frequently misdiagnosed as

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hypertensive nephrosclerosis. If post-transplant TMA develops in a younger patient where the

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underlying etiology of ESRD is not known, or recurs in the allograft or in subsequently transplanted kidneys, genetic testing to identify underlying complement regulatory defects

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should be considered to establish if the patient in fact has recurrent aHUS as opposed to post-

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transplant de novo TMA. This has important clinical ramifications; while treatment options for de novo TMA are limited, eculizumab, an anti-C5 monoclonal antibody that blocks the terminal

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complement pathway has been shown to be effective in treatment as well as prevention of

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recurrent aHUS after transplantation.[61]

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4. Treatment

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Diagnosis of post-transplant de novo TMA is associated with poor allograft and patient outcomes. Forty to 50 percent of patients lose their grafts within two years of diagnosis.[16, 18]

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The USRDS-based study by Reynolds et al published in 2003 reported patient mortality at 50 percent at three years after diagnosis. Several other studies report a 30 – 40% graft loss rate at 2 to 3 years after transplant [Table 2].[16-18, 32] Systemic TMA leads to early graft loss more frequently than localized TMA. In the study by Schwimmer et al, 54% of patients with systemic TMA developed dialysis requiring acute kidney injury and 38% had TMA-related graft loss. No patient with localized TMA required dialysis or experienced early graft loss. Despite this early separation in survival curves, long-term graft loss in the two groups was similarly poor.[16]

ACCEPTED MANUSCRIPT 16 As discussed above, post-transplant de novo TMA is a highly heterogeneous group with varied pathogenic mechanisms associated with different etiologies. Treatment is usually implemented

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in a step-wise fashion depending on what is believed to be the most likely etiology in a particular

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patient and how the patient responds to initial treatments. Various modalities and our

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proposed approach to treatment are discussed next [Figure 4]:

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4.A. CNI or mTOR inhibitor management: For patients where immunosuppressive medications are believed to be the cause of TMA, there are several case reports and series documenting

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resolution of TMA after switching from a CNI to another CNI or to an mTOR inhibitor.[17, 32, 6267] Satoskar et al, on the contrary, showed no difference in outcomes between ongoing use,

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temporary withdrawal, remodulation or discontinuation of cyclosporine in patients with de novo TMA.[18] There are no controlled studies that have prospectively evaluated effectiveness of this

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approach. Be that as it may, discontinuation of these potentially offending drugs is usually the

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parameters.

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first step in clinical practice and this frequently results in improvement in hematological

4.B. Plasma exchange (PLEX) and intravenous immonuglobulin (IVIG): Urgent PLEX has been shown to dramatically reduce mortality in patients with TTP[68, 69] and has long been used as first line treatment for aHUS as well (although, anti-complement therapy with eculizumab is now replacing PLEX for the treatment of aHUS given its better effectiveness and safety profile). This use of PLEX has been extrapolated to patients with post-transplant de novo TMA, especially those with systemic TMA with some success. Karthikeyan et al documented in 2003 in a series of 29 patients with de novo TMA diagnosed based on biopsy review, hemolytic anemia and thrombocytopenia that plasma exchange in addition to CNI withdrawal resulted in a graft

ACCEPTED MANUSCRIPT 17 salvage rate of 80 percent.[59] They speculated that this might be a result of removal of platelet aggregating factors such as thromboxane A2 and simultaneous replenishment of deficient

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factors such as PGI2-stimulating factor. With identification of complement regulatory

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abnormalities in kidney transplant recipients with systemic de novo TMA[19], it is plausible that in a subset of patients with acute TMA and underlying complement dysregulation, PLEX may

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lead to improved outcomes by providing normal functioning complement proteins and/or

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removing defective mutant proteins and/or auto-antibodies to complement factors.[19] Another recent study documented systemic TMA in 5 solid organ transplant recipients; response

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with no evidence of relapse was achieved in 100% of the patients with withdrawal of offending agent (tacrolimus or sirolimus) in all, PLEX in 4, eculizumab in 2 and rituximab in 1 patient.[67] In

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AMR-associated TMA, improved outcomes have been documented with PLEX and IVIG therapy,

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antibodies.[18, 70]

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presumably as a result of removal of and/or down-regulation of production of anti-donor HLA

4.C. Belatacept: Belatacept is a cytotoxic T-lymphocyte-associated antigen immunoglobulin

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(CTLA4-Ig) that blocks co-stimulatory interaction between CD80 and CD86 surface ligands on antigen presenting cells and CD28 on T cells. While listed as a treatment option for post— transplant TMA, it is important to note that belatacept itself does not treat the underlying endothelial injury. Rather, it is an effective immunosuppressive agent that allows discontinuation of CNIs or mTOR inhibitors that are believed to be toxic to the endothelium and are implicated in pathogenesis of post-transplant TMA. The first case report documenting successful use of belatacept for immunosuppression in a patient with post-transplant TMA associated with cyclosporine, tacrolimus and sirolimus was published in 2009; at nine months follow up, the patient had resolution of TMA with no adverse events.[71] Since then, two small

ACCEPTED MANUSCRIPT 18 case series documenting resolution of TMA and good graft outcomes after conversion from CNI to belatacept have been published.[72, 73] In patients with post-transplant TMA where

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immunosuppressive drugs are believed to be the offending agents, belatacept appears to be a

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promising alternative.

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4.D. Complement inhibition: Eculizumab, a recombinant, fully humanized hybrid IgG2/IgG4

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monoclonal antibody targeted against human complement protein C5 blocks generation of the lytic C5b-9 membrane attack complex. It has revolutionized the management of aHUS and has

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also been showed to be effective in the treatment and prevention of recurrent aHUS after transplantation.[61, 74] In post-transplant TMA, available data suggest that the complement

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system is activated in a large majority of patients, including those in whom offending mutations in complement regulatory genes are not identified. In a study by Chua et al, widespread renal

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deposition of C4d was observed in all histologically confirmed cases of post-transplant TMA;

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furthermore, C4d deposits co-localized with terminal complement complex C5b-9 deposits in most.[57] These data suggest that complement over-activation is one of the final common

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pathways in TMA in a heterogeneous group of patients. As such, anti-complement therapies likely have a role in management of de novo post-transplant TMA. There are several case reports and small case series that document effectiveness of eculizumab in kidney transplant recipients with severe medication-associated TMA that is refractory to previously mentioned treatments, including those where an underlying genetic detect is not identified. [67, 75-80] Similarly, there are also a few case reports where eculizumab was found to be effective in refractory AMR, with or without TMA [81-88]. A recently published small randomized clinical trial with 15 subjects with chronic-active AMR showed that 6 month of eculizumab treatment provided stabilization of renal function compared with the gradual

ACCEPTED MANUSCRIPT 19 decline noted in the observation group; patients with or without TMA were not specifically analyzed in this study.[89] On the contrary, in a study by Cornell et al that compared outcomes

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in eculizumab-treated positive crossmatch kidney transplants with a historical control group, the

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incidence of acute clinical AMR was lower in the eculizumab group, however, there was no difference in death-censored graft survival or biopsy findings at 1-year protocol biopsies.[90]

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Another trial evaluating efficacy of eculizumab versus PLEX and IVIG in AMR (ClinicalTrials.gov

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Identifier: NCT01895127) was terminated early reportedly due to lack of efficacy. Given these conflicting results and the expense of the drug, we recommend use of eculizumab in AMR-

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associated TMA where hemolysis persists despite maximal management including PLEX and in those with PLEX dependency. This drug does appear have a role in management of a subset of

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cases of de novo TMA. However, further work is needed to establish which kidney transplant recipients with this complication are most likely to benefit from eculizumab therapy and what

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5. Summary

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biomarkers of complement over-activation could be used to identify them.

The diagnosis of post-transplant TMA portends poor patient and allograft survival. De novo TMA after kidney transplantation comprises a large majority of the cases with post-transplant TMA, and represents a substantially more heterogeneous and complex entity than aHUS. Clinical presentation is highly variable, with laboratory findings of hemolytic anemia, thrombocytopenia and acute kidney injury present in less than half of the cases. Renal allograft biopsy remains the gold standard for establishing this diagnosis. First line therapy is usually withdrawal of offending agent such as CNI or mTOR inhibitor. Availability of alternative immunosuppressive medications such as belatacept and anti-complement therapies such as eculizumab provide encouraging

ACCEPTED MANUSCRIPT 20 options for use in these patients. However, further long-term data are needed to document

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efficacy of these treatments in patients with the diagnosis of de novo post-transplant TMA.

1. Recurrent TMA after transplantation

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Table 1: Classification of Post-transplant TMA

Atypical hemolytic uremic syndrome

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Thrombotic thrombocytopenic purpura

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Autoimmune disorders and glomerulonephritis with previously documented

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

TMA in native kidneys e.g. scleroderma and systemic lupus erythematosus, with

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or without anti-phospholipid antibodies

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2. De novo TMA after transplantation Immunosuppressive medication associated – TMA: Calcineurin inhibitor or

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mammalian target of rapamycin inhibitor or combination of the two Antibody-mediated rejection associated TMA

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Genetic, associated with complement regulatory gene abnormalities

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Medication related e.g. anti-vascular endothelial growth factor inhibitors

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Viral infections, e.g. hepatitis C, cytomegalovirus, parvovirus and BK

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C3 glomerulopathies as cause of ESRD, where phenotypical shift to aHUS after

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transplantation can occur 

Recurrent TMA, where a native kidney biopsy is not pursued and TMA as cause of ESRD is not established

ACCEPTED MANUSCRIPT 21 Table 2: Important clinical studies that have systematically evaluated incidence and risk factors for de novo TMA after transplantation Population/ database investigated

Methodology used to identify de novo TMA cases

Incidence of de novo TMA

Risk factors for development of de novo TMA

Outcomes and treatment options for de novo TMA

Reynolds et al[1] (2003)

USRDS database for transplants done between 1/1998-7/2000, with Medicare as the primary payer n=15,870

Medicare Claim for diagnosis of TMA based on International Classification of Diseases, 9th Revision Diagnosis Codes

0.8% (112) of kidney transplant recipients with non-HUS related ESRD; representing 75% of all cases of post-transplant TMA

- Younger recipient age - Older donor age - Female recipient -Initial use of sirolimus (? Confounding by indication)

- Patient survival 50% at 3 years after diagnosis - Graft survival not evaluated - Individual treatment options not evaluated

Langer et al [15] (2002)

2 cohorts of kidney transplants between 11/1993 – 10/2000 on cyclosporine/ sirolimus/ steroid regimen n=672

Systemic microangiopathic hemolytic anemia based on laboratory parameters

1.5% (10)

- High cyclosporine and sirolimus levels

Schwim mer et al [16] (2003)

Biopsy reports of kidney and kidney-pancreas transplants from 1985 to 2000 at a single center n=742

Histo-pathologic evidence of TMA

2.8% (21) - 62% (13) with systemic TMA, with hemolysis and thrombocytopeni a - 38% (8) with renal limited TMA

Not evaluated

Zarifian et al [17] (1999)

Kidney and kidney-pancreas transplant recipients from 1/1994 to 12/1996 at a single center n=188

Histo-pathologic evidence of TMA

13.8% (26) - 7.8% (8) with systemic TMA - 92.3% (24) with renal limited TMA

- Female recipient - Caucasian recipient - No difference in incidence of de novo TMA between living donor and deceased donor transplants

Satoskar et al [18] (2010)

Renal allograft biopsies from 1/200312//2008 at a single center n=958

Histo-pathologic evidence of TMA

6.1% (59); 55% with C4d positivity - 13.6% (33) with diffuse C4d positivity had TMA - 3.6% (26) without C4d positivity had TMA - 25% (15) with evidence of systemic TMA

- Antibody mediated rejection

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Study

- Recovered promptly after withdrawal of cyclosporine and sirolimus: 5 (2: previous regimen was successfully reinstituted 2: maintained on cyclosporine/ MMF/ prednisone 1: recurrent TMA after switch to tacrolimus, maintained on sirolimus/ prednisone) - Did not respond to discontinuation of cyclosporine but recovered after PLEX (6-16 times) +/- OKT3: 5 - Short term, 54% (7) of patients with systemic TMA required dialysis therapy and 38% (5) suffered TMArelated graft loss. - Long term graft survival was not different between systemic and localized TMA groups - Systemic TMA patients were treated more often with PLEX (38% vs 13%), although graft outcomes were similar among those who received PLEX vs those who did not. - 1 year graft survival: 81% - 3 year graft survival: 69% - Most common management strategy employed in 16 patients was replacement of cyclosporine with tacrolimus, with 13 functioning grafts at 1 year

- Graft loss within 2 years of biopsy with C4d-positive TMA: 40% (13/33) of patients with C4d positive TMA - Graft loss within 2 years of biopsy with C4d-negative TMA: 42% (11/26)

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Wu wt al (2016) [40]

Le Quintrec (2008) [19]

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Histo-pathologic evidence of TMA

Not analyzed

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Consecutive renal allograft biopsies routinely stained for C4d deposition over a period of 51 months (n=1073 with adequate tissue and clinical data from 563 allografts) Adult kidney transplant recipients at a single center with TMA and AMR (n=32) and those with TMA without AMR (n=31) Adult kidney transplant recipients from 5 centers who developed de novo posttransplant TMA were retrospectively recruited (n=24)

7.3% (36) - 19 were not drug related (AMR: 11, viral infection: 5, vasculitis: 1, recurrent HUS:1 ) - 17 were drug related (cyclosporineeverolimus: 6, tacrolimuseverolimus: 2, tacrolimus without mTOR inhibitor: 8, cyclosporine without mTOR inhibitor: 1) 3.4% (37)

- Graft loss reported in 30.7% (4/13)

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Histo-pathologic evidence of TMA

- Cyclosporine and sirolimus combination (RR 16.1 compared with tacrolimus and mycophenolate group) - Higher blood levels of immunosuppressive drugs. Sum of concentrations of cyclosporine and everolimus of the two drugs were 15.2±6.3 ng/mL in patients with TMA vs 10.7±2.1 ng/mL in patients without TMA (p<=0.034).

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3.5% (13) - 25% (3/13) with systemic TMA

- Graft loss was significantly greater in early C4d+, TMA+ group than C4d+ controls without TMA (57% vs 9.5%, p=0.02).

Not applicable

Antibody mediated rejection

- At 8-years post-transplant, death censored graft survival rate in the TMA+, AMR- group was 62.8% compared with 28% in TMA+,AMR+ group (p=0.01) - Patient survival was similar between the groups up to 8 years posttransplant

Not applicable

- Mutation in CFH or CFI gene in 29% (7/24); two of these had a mutation in both genes. On the contrary, no mutation was identified a 25 control kidney transplant recipients without TMA.

- Graft loss within first year was 33% (8/24) including 42% (3/7) in those with complement regulatory gene mutaton(s) and 29% (5/17) in those without.

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- Early (≤ 90 days) C4d+ biopsies were associated with more frequent TMA

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Meehan et al (2011)[3 9]

Histo-pathologic evidence of TMA in absence of vascular rejection

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Nava et al (2014) [36]

Kidney and kidney-pancreas transplant recipients from 1/1996 to 12/2002 n=368 For-cause renal allograft biopsy specimens gathered from 1998 to 2012 in 496 renal transplant recipients N=350

Histo-pathologic evidence of TMA

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Fortin et al [32] (2004)

Thrombocytopenia (platelets <100,000/mm3) and/or microangiopathic hemolytic anemia (Hemoglobin<10 g/dL) with LDH >2fold higher than normal values associated with a negative Coomb’s test and/or acute renal failure defined as plasma creatinine >120% of baseline. Patients with at least one of these criterion and

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histologic criterion of TMA were included.

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Figure 1: Proposed algorithm for diagnosis of post-transplant de novo TMA

Figure 2: Acute thrombotic angiopathy; glomerular abnormalities.

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Figure 2 (cont): Acute thrombotic angiopathy; arterial and arteriolar lesions.

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Figure 3. Chronic thrombotic angiopathy; glomerular and vascular abnormalities.

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Figure 4: Treatment of post-transplant de novo TMA

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