—  SPECIALTY CONFERENCE  —

Renal Pathology

Case 2 - Thrombotic Microangiopathy

Lorraine C. Racusen
Johns Hopkins School of Medicine
Baltimore, MD


Click on each slide thumbnail image for an enlarged view
Clinical History
The patient is an 18 year old male 11 months status post his second kidney transplant. He had developed end-stage renal disease by the age of 12 due to chronic tubulo-interstitial disease of unknown etiology. After about a year on peritoneal dialysis, the patient had received an initial living unrelated transplant from an altruistic donor. The graft was a 3-antigen match; the patient was CMV and EBV negative. The donor was CMV positive, and appropriate prophylaxis was given. A biopsy was performed 5 days post transplant for deterioration of renal function, and revealed focal thrombotic microangiopathy (TMA), with very focal infarction. Because of concern about possible tacrolimus toxicity, he was converted to Cellcept and prednisone, and eventually to cyclosporine. He subsequently had 2 biopsy-documented episodes of acute rejection, perhaps due to non-compliance, at 1 month and 13 months post transplant. Both rejections were mild vascular type, Banff type 2A, with a significant tubulo-interstitial component; immuno-staining for C4d was not being done at that time. He was treated with OKT3 and ATGAM for these rejection episodes. Chronic allograft dysfunction and fibrosis evolved in the graft, and he returned to dialysis. Due to recurrent fevers, rejection, and persistent requirement for immunosuppression, he underwent graft nephrectomy approximately 2.7 years post-transplant. Histological examination revealed acute and chronic rejection, with focal transmural arteritis and extensive fibrosis in vessels and parenchyma; no viral inclusions were detected.

The second allograft was a 6 antigen match from a deceased donor. He had thymoglobulin induction to avoid steroid use (he had avascular necrosis with involvement of right femoral head) and to avoid calcineurin inhibitors. He had initial good urine output, but only slow decline in serum creatinine. He developed fevers, migratory arthralgias, and decreased platelets and hemoglobin – testing for anti-donor antibody was repeatedly negative. At 12 days post-transplant, a biopsy revealed acute rejection, Banff type 2A, with diffuse capillary staining for C4d. He remained antibody negative, and he was treated with OKT3, with return of brisk urine output and fall in creatinine to 1.3 mg%. He was discharged on Rapamycin and Cellcept.

In the subsequent few months, he had a persistent lymphocele around the kidney, and required placement of drains and a nephrostomy tube. A biopsy at 4 months revealed infiltrates suspicious for rejection, with mild capillary margination and diffuse capillary staining for C4d (1-2+); antibody screens remained negative. At 6.3 months post transplant, he presented with fever, abdominal pain, increased drain output and graft tenderness. Open renal biopsy revealed acute rejection, Banff type 2B, superimposed on chronic rejection. Neutrophil margination was noted in capillaries, with "fairly diffuse" staining for C4d (1-2+). Approximately 9 months post transplant, he was hospitalized to place a JP drain in the perinephric lymphocele; culture of fluid revealed VRE, treated with antibiotics. Renal biopsy revealed acute rejection, Banff type 2A, with moderate evolving chronic changes and neutrophils in glomerular and peritubular capillaries – R/O anti-donor antibody; screens for anti-donor antibody remained negative. The patient had been chronically hypertensive on multiple medications. He was switched from Rapamycin to cyclopsporine, due to marrow suppression. At 11 months post-transplant, he developed a lung infiltrate and required ventilator support. His immunosuppression was stopped, and he developed a clinical acute rejection. Allograft nephrectomy was performed.


Case 2 - Figure 1 - Low power view of section from allograft nephrectomy, showing 2 glomeruli, 1 ischemic and 1 with microangiopathic changes, including "bloodless" capillaries with flocculent material filling capillary loops and enmeshed erythrocytes with fragmentation. Background with interstitial edema, fibrosis and acute tubular injury.
Case 2 - Figure 2 - Higher power view of a "bloodless" glomerulus with swollen endothelial cells, and entrapped focally fragmented erythrocytes.
Case 2 - Figure 3 - Artery with marked intimal expansion, with myofibroblasts and a few entrapped mononuclear cells. Note endothelial activation but no arteritis.



Case 2 - Figure 4 - Artery with neo-intima formation and mononuclear inflammatory cells in middle layers of the thickened intima, findings of chronic rejection.
Case 2 - Figure 5 - Area of medullary hemorrhage and capillary congestion.
Case 2 - Figure 6 - Cells marginating in peritubular capillaries, including neutrophils and mononuclear cells. Note focal capillary endothelial activation.



Case 2 - Figure 7 - Isometric vacuolization in tubular cells, with apical blebbing. Some marginating cells in peritubular capillaries.
Case 2 - Figure 8 - Small vessel angiopathic changes, with thrombus and entrapped erythrocytes in vessel walls. Reactive and regenerative tubular cell changes.
Case 2 - Figure 9 - Focal peritubular capillary staining for C4d (1-2+) involving about 15% of capillaries overall.

Diagnoses/Differential Diagnosis:
The major finding in this kidney, in addition to chronic rejection and acute tubular injury, is thrombotic microangiopathy. There are numerous "bloodless" glomeruli filled with flocculent material in capillaries, with some severe capillary congestion and focal fragmentation of erythrocytes, and focal thrombosis and mural injury with extravasation of erythrocytes in arterioles. Focal peritubular capillary congestion and margination of inflammatory cells, including neutrophils, is noted. In addition, there is a small infarct present on at least some of the sections. Finally, focal isometric vacuolization can be identified in tubular epithelial cells. Arteries have focally severe fibrointimal thickening, with mild edema and lymphocytes present deeper in the intima is some arteries, consistent with chronic rejection. Immunostaining for C4d reveals trace-1+ staining in about 15-20% of peritubular capillaries.

Discussion:
Possible causes of thrombotic microangiopathy (TMA) in the renal allograft include a variety of processes. In the setting of the renal allograft, the most likely causes include:

  1. recurrent (or de novo) hemolytic uremic syndrome/TTP;
  2. drug toxicity, including especially calcineurin inhibitors;
  3. antibody-mediated rejection;
  4. anti-phospholipid antibody syndrome/autoimmune disease; and
  5. malignant hypertension/scleroderma
The morphology of these entities is similar, not surprising since they all result from endothelial injury, tipping the normal balance between thrombotic and anti-thrombotic factors in the microvasculature toward thrombosis. Platelet activation and capillary injury can be demonstrated in a variety of processes in the renal allograft [1]. Vascular changes in malignant hyptertension/scleroderma tend to be a bit different, with more arterial involvement and more fibrinoid necrosis of vessel walls, features not seen in this case. Therefore, while the patient did have persistent hypertension which could have contributed to endothelial injury, this is probably not the primary insult, and the discussion will focus on the other entities.

In a recent analysis of incidence, time to event and risk factors for TMA in the allograft, the USRDS data base was utilized to identify a historical cohort of recipients from January 1, 1998 to July 31, 2000, followed until December 31, 2000. Among those with end-stage renal disease due to HUS, 29.2% had post-transplant TMA versus 0.8% incidence in other patients. Risk was highest in the first 3 months post-transplant, but occurred later as well. Risk factors for de novo TMA included younger recipient age, older donor age, female recipient and initial use of sirolimus. Patient survival rate after TMA was approximately 50% at 3 years [2]. Occasional cases of infection temporally related to de novo post-transplant TMA have been reported, including CMV [3] and hepatitic C [4].

Certain forms of HUS recur more frequently post-transplant. In a recent review of the literature, among 118 children transplanted after post-diarrheal HUS due to toxin-induced endothelial injury, only 0.8% had recurrence. In contrast, of 63 children with diarrhea-negative HUS of unknown mechanism, 21% had recurrence post-transplant. Of those with known underlying mechanisms of HUS, recurrence and graft loss were highest in those with factor H deficiency, and low serum C3. The only patient with constitutional deficiency of von Willebrand factor-cleaving protease, an underlying mechanism of thrombotic thrombocytopenic purpura (TTP), had recurrence and graft loss [5]. Another recent study in pediatric patients also documented no recurrence and excellent outcome in 66 patients with ESRD due to Shiga-toxin-induced HUS [6]. Autosomal recessive and dominant forms of HUS have high recurrence rates. Progress in the understanding of the mechanisms and genetics of diarrhea-negative HUS are needed to more accurately predict recurrence rate and therapeutic approaches in this cohort.

Calcineurin inhibitors (CNIs) not uncommonly produce some diminution of GFR. Even therapeutic doses of CNIs can activate endothelial cells, causing release of vasoactive factors balanced toward vasoconstriction, such as endothelin and thromboxane, resulting in some "functional" reduction of GFR, without morphological renal injury and rapidly reversible on discontinuation of or reduction in dosage of the drug. At high doses and/or in sensitive individuals, more severe endothelial injury with release of Von Willebrand factor multimers and platelet activating factor and features of hemolytic uremic syndrome may develop [7, 8] . Incidence of TMA has been reported to affect 1-14% of renal transplant recipients administered CNI-based immunosuppression [9]. Initially reported with cyclosporine, especially when used in high doses and peaking again with introduction of the micro-emulsion formulation [8, 10, 11, 12] , this complication has also been reported with therapy with tacrolimus (Prograf) [7, 13, 14, 15] . It is sometimes possible to reduce or discontinue one agent and re-introduce the same or alternative CNI at a later time [11, 12] , though this is not without risk [16]. The availability of other agents makes it possible, as in this case, to avoid CNI use completely in many patients. Arterioles are a primary target, with endothelial injury and/or ischemia in glomeruli. There is often isometric vacuolization in tubular epithelial cells, a fairly reliable sign of exposure to high-dose calcineurin inhibitors. CNI are generally used with caution in patients at risk for TMA recurrence in the allograft.

TMA has also been reported in patients being treated with other immunosuppressive agents, including OKT3 [7, 17] . Potentiation of CsA-induced TMA has been reported with contemporaneous or contiguous CNI and sirolimus use, perhaps due to increased intrarenal CsA levels in this setting [18, 19] . A few cases of TMA in patients treated with sirolimus without CNI or OKT3 have also been reported (e.g. 20)

Small vessel thrombosis and necrosis are also among the morphological correlates of antibody-mediated/severe vascular rejection in the allograft [21, 22] . In these cases, endothelial injury apparently results from engagement of anti-donor antibody with antigens expressed on endothelial cells, including HLA antigens (class I and II), AB antigens, and occasionally other endothelial antigens, causing complement engagement and activation, with triggering of the complement cascade, recruitment of inflammatory cells, and damage to endothelium and vessel wall if severe. Morphological features in some cases include fibrin thrombi in glomeruli or vessels, or thromboses in glomeruli or arteries. Other more common signs of antibody-mediated rejection, including margination in capillaries of neutrophils and mononuclear cells/monocytes, arteritis, and immunostaining for C4d in a linear along peritubular capillaries, provide morphological clues that AMR is present in the allograft [23]. In the absence of these features and/or demonstration of anti-donor antibody, other causes of endothelial injury should be considered.

Anti-phospholipid antibody syndrome can recur post-transplant [24], sometimes in the setting of systemic lupus erythrematosus, resulting in increase in increased risk of graft thrombosis [25]. Morphology is very similar to other forms of TMA, and diagnosis is made by demonstration of the antibody in the patient's serum. Renal TMA has been reported in HCV+ patients with anti-cardiolipin antibody post-transplant [4].

Clinical presentation in patients with post-transplant TMA typically includes acute renal dysfunction, often with hematological findings as well. In a recent series, 12/21 affected recipients had "systemic" TMA with hemolysis and thrombocytopenia, whilc 8 patients had TMA apparently localized to the allograft. The former cohort had more severe dysfunction and higher rate of graft loss [9]. Withdrawl or treatment of precipitating factors is the most effective therapeutic approach. Sirolimus has been proposed as a CNI-sparing alternative in patients with drug-induced HUS, though potentiation of CNI toxicity has occasionally been reported [17]. Plasmapheresis/plasma exchange has been used to attempt to limit the process. While general efficacy has not been established [26], some series report good outcomes using these strategies [27] . Outcome is variable, depending on the underlying cause and severity; as noted above, outcome in recurrent forms is generally poor.

In the current case, the native kidney disease was documented by biopsy; there was no evidence of thrombotic microangiopathy in the native kidney, so that recurrence is not an issue in this case. There were biopsy findings suggestive of CNI toxicity at 5 days in a first allograft, with no evidence of rejeciton; calcineurin inhibitors were discontinued following that early biopsy, and not re-introduced while that allograft was in place. CNI were also avoided in the second allograft, until weeks prior to allograft nephrectomy. Recurrent rejection episodes (Banff type 2A) in the first allograft were ascribed to non-compliance. In the second allograft, there were documented acute rejection episodes, Banff type 2a, with persistent morphological features suggestive of AMR and positive immunostaining for C4d without any anti-donor HLA antibody ever demonstrated during the patient's course. This, of course, does not rule out anti-donor antibody not detectable by routine screening methods. There are a number of potential explanations for failure to demonstrate anti-donor antibody during AMR. One is that methods of detection being used are not sensitive enough – this is generally not a problem with modern detection methods, but occasionally is relevant in deceased donor transplants, since there may be time only for cytotoxic cross-match and not for flow cross-matching pre-transplant, so that pre-existing low levels of anti-donor antibody may be missed. Another possibility is that the antibody is non-HLA; more sophisticated screening against a panel of endothelial antigens is possible, but was not performed in this case. In addition, it is possible that antibodies were present episodically and/or at very low levels, and were adsorbed to the graft; the clinicians were encouraged to re-screen the patient's serum post-nephrectomy, in hopes of capturing an antibody peak on removal of the allograft. Only focal fairly weak staining for C4d was present in peritubular capillaries at nephrectomy. There was no evidence of anti-phospholipid antibody post-nephrectomy.

The final pathology in this nephrectomy specimen is most consistent with HUS, probably induced by re-exposure to CNI in the weeks prior to nephrectomy, superimposed on a background of chronic rejection.

References

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