Renal Thrombotic Microangiopathy after Hematopoietic Cell Transplant: Role of GVHD in Pathogenesis

Materials and Methods

Histologic Methods and Definitions of TMA

Renal pathology from 322 patients was reviewed by a renal pathologist (S.C.) who was blind to the clinical information of each case. All positive cases and a sample of negative cases were reviewed by a second renal pathologist (C.E.A.). Two sections of renal tissue from each patient, one stained with hematoxylin-eosin and a second stained with periodic-acid Schiff, were reviewed. Renal histologic findings were classified into three groups: (1) no renal thrombosis; (2) typical TMA morphology which includes fibrin thrombi within glomeruli and mesangiolysis, and/or thrombosis involving arterioles or interlobular arteries in conjunction with characteristic intimal swelling in these vessels of the subendothelium and occlusion of capillary loops (Figure 1); and (3) isolated thrombosis, defined as a single glomerular or vascular thrombus without typical TMA morphology (Figure 2).

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Figure 1.

Thrombotic microangiopathy involving a glomerulus. There are multiple thrombi in capillary loops, (arrows), segmental mesangiolysis(double arrows) and duplication of basement membranes in some capillary loops (arrowheads). Periodic-acid Schiff stain at 40× magnification.

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Figure 2.

Glomerulus showing isolated intracapillary thrombus but with otherwise normal glomerular tuft architecture. Periodic-acid Schiff stain at 40× magnification.

We defined clinical evidence of TMA in two ways to investigate the relationship between clinical parameters in the two weeks before death and histologic findings at death. Definition 1 is hematocrit < 30, platelet count < 100,000, LDH above the upper limits of normal, and a 50% increase in the baseline serum creatinine, in a single day's measurements. Definition 2 is hematocrit < 30, platelet count < 100,000, LDH above the upper limits of normal, and a 50% increase in the serum baseline creatinine, each occurring at least once over the two weeks’ measurements. LDH upper limits of normal are defined as LDH > 425 U/L if less than 10 yr of age, LDH > 350 if age 10 to 15 yr old, and LDH > 250 if 15 yr or older. Medical charts were reviewed before death to determine whether the patients carried a clinical diagnosis or suspicion of TMA. Autopsy reports were reviewed to determine the cause of death.

Results

Demographic data and variables identified as potential risk factors for development of TMA or an isolated thrombus are presented in Table 1. The mean age of the cohort was 42.7 yr, with a range of 6 mo to 68 yr. Sixty-four percent of patients received TBI as part of their conditioning regimen before transplant. Sixty-five percent of patients developed acute GVHD grades II to IV after transplant. Forty-one percent of patients developed AKI after transplant, and the mean serum creatinine in the two weeks before death was 1.56 mg/dl. Fifty-nine percent of patients developed a viral infection, 33% a bacterial infection, and 11% a fungal infection after transplant and before death.

Patients with negative histology developed AKI at a median of 15 d post-HCT (range 1 to 74), whereas patients with histologic evidence of TMA or an isolated thrombosis developed AKI later, at a median of 32 d (range 0 to 100) and 27 d (range 7 to 95) post-HCT, respectively. Twenty-six percent of all patients required dialysis during the two weeks before death.

Discussion

We found that acute GVHD grades II to IV, patient–donor sex mismatch (female patient and male donor), TBI > 1200 cGY, and ADV infection were associated with an increased risk of TMA in the kidney at autopsy. Our findings are similar to those of studies of TMA based on clinical criteria alone (8–10). The frequency of TMA in this cohort of patients was 20% compared with a frequency of 0.5% to 63.6% reported in the literature, whether defined solely by clinical criteria or by histology as in this cohort (11). Our data suggest, however, that there is little correlation between TMA as diagnosed by clinical criteria and histologic evidence of TMA in the kidney.

Although calcineurin inhibitors such as cyclosporine are frequently implicated as the cause of TMA in patients after transplant, cyclosporine use did not contribute to histologic evidence of TMA in this cohort of patients.

Endothelial injury in the kidney may be secondary to circulating inflammatory cytokines related to GVHD elsewhere in the body or may reflect direct injury to endothelial cells of the kidney by GVHD. Endothelial injury has been described in patients with chronic GVHD, and it is thought that endothelial cells are direct targets of cytotoxic donor T lymphocytes (12). Plasma markers of endothelial injury and coagulation activation are elevated in patients with acute GVHD after HCT, suggesting an association between endothelial injury, acute GVHD, and the subsequent development of TMA (13,14). Newer evidence suggests a role for vascular endothelial growth factor (VEGF) in the development of TMA and GVHD (15,16). Inhibition or loss of function of VEGF leads to development of proteinuria and glomerular endothelial injury consistent with TMA (15), and lower levels of VEGF have been associated with more severe forms of acute GVHD (16). GVHD may lead to endothelial injury in the kidney through a reduction in serum VEGF levels and therefore loss of the protective effects of VEGF on the glomerular filtration barrier. Thus, TMA in the kidney may be a manifestation of renal effects of GVHD. Our finding of patient–donor sex mismatch as a significant risk factor further supports the idea that acute GVHD plays a role in TMA after HCT because these patients are at increased risk for acute GVHD (17,18). The increased risk of GVHD may be related to the selective expression in females of minor histocompatibility antigens on the X-chromosome that are not expressed in males. These minor H antigens may then be recognized by T cells after transplant in females who receive cells from a male donor (17,18).

The significance of a single thrombus is unclear. Its presence may be an early manifestation of TMA or a low-grade TMA. It may be that those patients who develop only a single thrombus have a less severe grade of GVHD. Of course, the finding of a single thrombus may also be due to sampling.

Although elevations in serum creatinine are part of the definition of TMA, we did not find that the histologic changes in the kidney were reflected in serum creatinine measurements in the two weeks before death. This suggests that significant subclinical renal injury occurs early after transplant and that serum creatinine does not accurately measure kidney function in patients with mild renal insufficiency, or in certain other patient populations (e.g., individuals with malnutrition, muscle wasting, cancer, or the elderly) (19,20). Patients undergoing hematopoietic cell transplant may have large fluctuations in their nutritional status, muscle mass, and weight that can influence serum creatinine levels. Only 119 patients (38%) had laboratory data available in the 2 wk before death that could be used to make a clinical diagnosis of TMA, in part because LDH is not part of routine laboratory testing in our patients and is usually drawn only when clinically indicated. However, among patients with LDH measurements, laboratory data did not correlate with a histologic diagnosis of TMA. In the study by Eremina et al. described above, patients also did not have serologic evidence of TMA at the time of biopsy but did have proteinuria (15). In addition, a clinical diagnosis of TMA in the patient's chart correlated with a histologic diagnosis of TMA in approximately one third of the cases. Therefore, a high index of suspicion for the development of TMA is needed in patients after HCT especially those with gender-mismatched donors, TBI > 1200cGy as part of their conditioning regimen, ADV infection, and acute GVHD.

Both TBI and CMV infections are commonly implicated in the development of TMA after transplant (21–25). TBI dose > 1200 cGY was also associated with TMA; however, the effects of TBI were confounded by donor type. In our study, ADV infection rather than CMV infection was associated with an increased risk of TMA. A previous case report has described ADV in a patient mimicking clinical TMA; however, the histologic findings at autopsy in the kidney did not demonstrate TMA (26). In a study of 21 patients from our institution, ADV nephritis was associated with acute kidney injury and diagnosed at autopsy (19 patients) or on biopsy (2 patients) by the presence of virus in the kidney. There was no histologic evidence of TMA in any of these patients (27). We found no evidence that bacterial or fungal infections in the 2 wk before death contributed to the development of TMA after transplant.

In this study, we found no evidence for an association between the average daily dose of cyclosporine and the presence of TMA in the kidney. Martinez et al. similarly found no association between cyclosporine plasma levels and clinical diagnosis of TMA in patients after HCT (28). These findings contradict other studies in which both cyclosporine and tacrolimus have been associated with clinical TMA in the HCT population (24,29–34). Recent reports have also found an association between sirolimus and development of TMA after HCT (35). It may be that our patients died earlier than others and that the duration of therapy of calcineurin inhibitors may be more important for endothelial injury than the cumulative dose.

There are limitations to applying the findings from an autopsy cohort to the general HCT population. Selection biases are inherent as patients who die are obviously sicker than those who survive. Not all patients undergo an autopsy, and even among those who do, the kidney may not always be preserved because autopsies may be limited to certain organs. Most patients who died at home or at an outside institution were not included in this study. Patients in this study died at a median of 43 d after HCT, and risk factors for TMA early after transplant may differ from factors that contribute to the later development of TMA. Finally, we did not evaluate tissue using electron microscopy, which may demonstrate more subtle changes of TMA. Among the strengths of this paper are that the pathologist reviewing the slides was blinded to any clinical information and thus was not biased in her characterizations, and the diagnosis of TMA was made on histologic grounds rather than clinical data, which can often be confounded by other disease processes.

We found that TMA was present in 20% of kidneys at autopsy in patients after HCT and that an additional 15% had evidence of thrombus formation. The risk of TMA is increased four-fold in patients with acute GVHD and is independent of cyclosporine use. We found no evidence that dose of cyclosporine contributes to the development of histologic TMA in the kidney. Therefore, stopping or decreasing the dose of cyclosporine as recommended in many clinical practice guidelines may not be helpful in managing TMA. Rather, continuing treatment of the acute GVHD or directing therapy to mitigate endothelial injury may be of benefit. Although obtaining renal biopsies may not always be practical, they are important for an accurate diagnosis. It is also important to monitor patients with acute GVHD for ongoing and persistent renal damage and urinary abnormalities that can persist after clinical resolution of TMA.

Disclosures

None.