Chronic Active Thrombotic Microangiopathy in Native and Transplanted Kidneys

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1 Available online at Annals of Clinical & Laboratory Science, vol. 36, no.3, Case Reports: Chronic Active Thrombotic Microangiopathy in Native and Transplanted Kidneys Ping L. Zhang, 1 Jeffery W. Prichard, 1 Fan Lin, 1 Michael F. Shultz, 2 Sayeed K. Malek, 3 John H. Shaw IV, 4 and James E. Hartle 2 1 Division of Laboratory Medicine, 2 Department of Nephrology, 3 Department of Transplantation, and 4 Weis Center for Research, Geisinger Medical Center, Danville, Pennsylvania Abstract. We report 2 complicated cases of thrombotic microangiopathy with chronic features and active components. The first case was a 36-yr-old woman with positive anti-dna antibody and possible lupus cerebritis, who developed thrombotic microangiopathy secondary to a series of syndromes, including preeclampsia and anti-phospholipid antibody syndrome. Renal biopsy revealed no evidence of lupus nephritis and her renal function returned to normal 1 week after the biopsy. The second case was a 46-yrold man who developed thrombotic microangiopathy of unknown etiology, which led to end-stage renal disease within 6 mo. The patient received a living related-donor transplant, but thrombotic microangiopathy recurred in the donor kidney only 40 days after the renal transplantation. Keywords: thrombotic microangiopathy, thrombocytopenia, preeclampsia, anti-phospholipid antibody syndrome, renal transplantation Introduction Thrombotic microangiopathy in the kidney usually develops secondary to hemolytic uremic syndrome, antiphospholipid antibody syndrome, toxemia of pregnancy, thrombotic thrombocytopenic purpura, or disseminated intravascular coagulation [1]. When no obvious intravascular thrombi are seen in the renal biopsy, it may be difficult to distinguish thrombotic microangiopathy from native glomerular diseases, such as membranoproliferative glomerulonephritis or chronic transplant nephropathy, based on pathologic changes in glomeruli. Here we report 2 cases of chronic active thrombotic microangiopathy; 1 case had several conditions related to thrombotic microangiopathy, and the other case involved recurrent thrombotic microangiopathy in a living related-donor kidney following renal transplantation. Address correspondence to Ping L. Zhang, M.D., Ph.D., Laboratory Medicine, Geisinger Medical Center, 100 North Academy, Danville, PA 17822, USA; tel ; fax ; plzhang@geisinger.edu. Case Reports Case 1. The patient was a 36-yr-old pregnant woman with no significant prior medical history. At 18 wk of gestation, she developed thrombocytopenia and hypertension (189/110 mm Hg). After a seizure occurred at home, she was diagnosed as preeclampsia/eclampsia. At 19 wk of gestation, she had an elective abortion because of worsening preeclampsia. Pathologic examination of the placenta was not remarkable. One week after the abortion, she continued to have high blood pressure (173/94 mm Hg) and developed acute renal failure. Laboratory tests showed a positive antinuclear antibody (>100 dilution) and anti-double stranded- DNA (20 titer; normal range <10 titer). Anticardiolipin antibody titer was >100 GPL U/ml. Urinalysis showed proteinuria (100 mg/dl) and severe hematuria. Serum C3 and C4 concentrations were within normal ranges. Positive blood test results included prolonged activated partial thromboplastin time (APTT) (44 sec; normal 20 to /06/ $1, by the Association of Clinical Scientists, Inc.

2 320 Annals of Clinical & Laboratory Science, vol. 36, no.3, 2006 sec), prolonged lupus-sensitive APTT (69 sec; normal 31 to 53 sec), high D-dimer level (3.81 µg/ ml; normal <0.50 µg/ml), elevated serum alkaline phosphatase activity (157 U/L; normal 25 to125 U/ L), and elevated serum aspartate aminotransferase activity (73 U/L; normal 8 to 46 U/L). A renal biopsy was performed for pathologic examination. Case 2. The patient was a 47-yr-old man who developed proteinuria (10 g/day) and microscopic hematuria (with normal serum levels of C3 and C4, and negative serology) at 1 yr before renal transplantation. Serological tests were negative for C3, C4, anti-dna antibody, anti-glomerular basement membrane antibody, and hepatitis C antibody. A native kidney biopsy was done and a diagnosis of chronic active thrombotic microangiopathy with some features of type I membranoproliferative glomerulonephritis was made. At that time, immunofluorescent histological staining for IgM was strongly positive; IgG staining was weak and C3 staining was negative. Subendothelial deposits were identified ultrastructurally and mesangial interposition was prominent. The interstitial fibrosis was mild in the native renal biopsy. The patient showed rapid deterioration of renal function with serum creatinine concentration increasing from 2 to 8 mg/dl and progression to end-stage renal disease within 6 mo. One year later, the patient received a living related-donor kidney transplant from his 18-yr-old daughter. Campath-1H (30 mg, iv) was given intraoperatively to induce tolerance, followed by post-operative immunosuppression. After transplantation, the patient s serum creatinine level dropped dramatically. A protocol renal biopsy performed at 1 wk post-transplantation was unremarkable in the glomeruli and the tubules except for a focal mildly thickened artery; there was no evidence of acute rejection. One month later, the patient s serum creatinine increased and he developed hematuria and proteinuria (>300 mg/ l). Three renal biopsies were subsequently performed because of declining renal function. Methods Frozen sections of renal biopsies were cut for direct immunofluorescent stains of IgG, IgA, IgM, C3, C1q, kappa, lambda, and albumin, using an autostainer (Dako Corp., Carpinteria, CA, USA). The remaining renal tissue was divided into 2 parts and fixed with 10% formalin and 3% glutaraldehyde, respectively. The formalin-fixed tissue was embedded, sectioned, and stained with hematoxylin-eosin (H&E), periodic-acid Schiff (PAS) stain, and Masson s trichrome stain for light microscopy. The tissue for electron microscopy was postfixed in osmium tetroxide, embedded in resin, sectioned, and stained with uranyl acetate and lead citrate. Grids for electron microscopy were examined using a transmission electron microscope (JEM-1200EX electron microscope, JEOL Co., Tokyo, Japan). Results Case 1. The serial changes of red blood cell count, platelet count, and renal function tests in this patient are listed in Table 1. By light microscopy, 9 glomeruli were identified in the renal biopsy; 3 of Table 1. Time-course of laboratory test results in Case 1. Interval before and Blood RBC Blood platelet Serum urea Serum Glomerular after renal biopsy count count nitrogen creatinine filtration rate (10 6 /µl) (10 3 /µl) (mg/dl) (mg/dl) (ml/min, estimated) 17 days > days* > days days biopsy day days days days days * Day of abortion

3 Case reports: thrombotic microangiopathy 321 the glomeruli were globally sclerosed. The remaining glomeruli showed lobulated features with mesangiolysis and fragmented red blood cell debris (Fig. 1A). Glomerular loops were congested with red blood cells and with thickened capillary loops (Fig. 1B). One glomerulus showed a ruptured Bowman s capsule, but definite crescent formation was not identified. The interstitium showed mild to focally moderate fibrosis and moderate lymphocytic interstitial nephritis in 1 of 3 biopsy fragments. The vessels were mildly thickened, but no vascular necrosis, vasculitis, or thrombosis seen in any vessel in the H&E, PAS, or trichrome-stained sections. Immunofluorescent studies of 3 glomeruli showed granular capillary loops and mesangial staining for fibrinogen (3+) (Fig. 1C), IgM (2+) (Fig. 1D), IgG (1+), IgA (+/-), C3 (2+), kappa (1+), and lambda (1+); staining for Ciq was essentially negative. No fibrinogen staining was noted in the vessels. Five glomeruli were examined by electron microscopy. There was segmental fusion of foot processes. Glomerular endothelial swelling (endotheliosis) and lipid vacuoles were noted (Fig. 1E). The glomerular basement membranes (GBM) revealed double contour with subendothelial expansion (Fig. 1F). Mesangial and paramesangial electron dense deposits were present, most likely being fibrin deposits (Fig. 1F). No deposits were seen at epimembranous locations. The proximal tubules were flat, with sloughed villi, consistent with acute tubular injury, but no deposits were seen along tubular basement membranes. No tubuloreticular inclusions were seen in the endothelium. Case 2. Light microscopic and electron microscopic findings in the native kidney biopsy are illustrated in Fig. 2A and 2B. The patient s initial protocol renal biopsy at 1 wk post-transplant was unremarkable. On day 29 following renal transplantation, a second biopsy was conducted because of the patient s deteriorating renal function (Table 2). This biopsy showed no acute cellular rejection; tubules were slightly dilated with diminished brush borders, consistent with mild acute tubular injury. Immunofluorescent stains were all essentially negative. Electron microscopy showed focal irregular thickened and undulating glomerular loops. This might be related to ischemic changes or Table 2. Time-course of renal function tests in Case 2. Interval Serum urea Serum Glomerular after renal nitrogen creatinine filtration transplantation (mg/dl) (mg/dl) rate (ml/min) (estimated) Pre-transplant Day of transplant Day Day Day 6 (Biopsy 1) Day Day Day 29 (Biopsy 2) Day 40 (Biopsy 3) Day 52 (Biopsy 4) Day Day early features of chronic transplant glomerulopathy, since no subendothelial deposits were seen; the villi were maintained and the patient did not have proteinuria or hematuria at that time (Fig. 2C). One week later (on day 40), the patient s serum creatinine level continued to be elevated and a third post-transplant biopsy was performed. No acute cellular rejection was evident on the permanent sections. A thrombus in an arteriole was seen, raising the possibility of thrombotic microangiopathy (Fig. 2D), although the patient s platelet count was normal. Twelve days later (on day 52), the patient developed proteinuria (>300 mg/dl) and moderate hematuria with abnormal urine sediment. Renal function tests were markedly abnormal (Table 2). A fourth post-transplant renal biopsy was performed, which showed prominent lobular features with thickened glomerular loops, mesangial expansion, and mild hypercellularity. Immunofluorescent examinations showed dominant staining of IgM, C3, and kappa, with weak staining for IgG, IgA, lambda, and C1q. Electron microscopy showed duplication of GBM and scattered subendothelial deposits. These findings were similar to those previously seen on the patient s native kidney biopsy. In addition, one artery was prominently thickened with onion-skin appearance. Overall, the findings were most consistent with recurrent thrombotic microangiopathy.

4 322 Annals of Clinical & Laboratory Science, vol. 36, no.3, 2006 Fig. 1. Morphologic changes of thrombotic microangiopathy in Case 1: (A) lobulated glomerulus with features of mesangiolysis and red blood cell debris (H&E x400); (B) congested capillaries and double track of glomerular basement membranes (PAS x600); (C) positive fibrinogen immunofluorescent staining of GBM (x1000); (D) positive IgM immunofluorescent staining along GBM and mesangial areas (x400); (E) endotheliosis and lipid vacuoles in endothelium causing occlusion of the glomerular capillary loop (electron microscopy x2,500); and (F) Duplication of glomerular basement membranes and fibrin deposits (possible mural microthrombi) (electron microscopy, x2,500).

5 Case reports: thrombotic microangiopathy 323 Fig. 2. Morphologic changes of thrombotic microangiopathy in native and transplant renal biopsies in Case 2: (A) lobular features of glomerulus with double contours of glomerular basement membranes in the native biopsy (PAS, x600); (B) duplication of glomerular basement membranes and edematous endothelium, leading to narrowed lumen in the native biopsy (electron microscopy, x2,500); (C) focal duplication of glomerular basement membranes, possible early features of chronic transplant glomerulopathy or early thrombotic microangiopathy in the second post-transplant biopsy (electron microscopy, x3,000); (D) arteriolar thrombus in the third post-transplant biopsy (trichrome, x400); (E) lobulated glomerulus in the last biopsy, similar to the native biopsy (PAS, x600); and (F) duplicated glomerular basement membranes with small electron-dense deposits, similar to the native biopsy (electron microscopy, x2,500).

6 324 Annals of Clinical & Laboratory Science, vol. 36, no.3, 2006 Discussion Although no definite thrombi were identified in Case 1, the findings of mesangiolysis, fibrinogen staining of glomerular capillary loops, and duplication of glomerular basement membranes were consistent with thrombotic microangiopathy. The findings of ultrastructural features of endotheliosis and lipid vacuoles supported residual components of preeclampsia. In contrast, absence of full house pattern of immunoreactive staining, particularly negative C1q staining, suggested no currently active lupus nephritis, although the patient had serological evidence of clinical lupus. This patient s chronic active thrombotic microangiopathy may reflect multiple etiologies. Since she had positive lupus sensitive APTT and anti-phospholipid antibody syndrome, her thrombocytopenia may have been related to lupusassociated coagulation abnormalities. In addition to the high blood pressure during her pregnancy and shortly following abortion, the presence of the mild elevation of serum bilirubin, high serum levels of liver enzymes, and low platelet count indicates that she might have developed pregnancy-related HELLP syndrome (hemolysis, elevation in liver enzymes, and low platelets). Furthermore, the high level of D-dimers suggested an early feature of disseminated intravascular coagulation. In summary, this patient s chronic active thrombotic microangiopathy, which occurred 10 days after fetal loss, could have been secondary to antiphospholipid antibody syndrome or preeclampsia, or a combination of these processes; the former may be the dominant etiology, whereas the latter may be a minor factor under such a clinical scenario. Thrombotic microangiopathy and toxemia of pregnancy (preeclampsia, eclampsia, and HELLP) were classified in different categories in the past [2-5]. A recent American Force Institute of Pathology (AFIP) fascicle has grouped toxemia of pregnancy in the thrombotic microangiopathy chapter [1], although the endotheliosis and lipid vacuoles seen ultrastructurally are unique features of toxemia of pregnancy involvement of the kidney [6]. In the native renal biopsy of Case 2, it was difficult to distinguish a thrombotic microangiopathy from a type I membranoproliferative glomerulonephritis, since (a) duplication of glomerular basement membranes can be seen under both situations; (b) substantial subendothelial immune complex deposits were present; and (c) there were no identifiable thrombi or clinical history of coagulation abnormalities in this patient. The signs that led us eventually to favor thrombotic microangiopathy in the native biopsy were that (a) IgM staining was stronger than IgG staining and (b) the features of recurrent thrombotic microangiopathy were nearly identical to the changes seen in the native biopsy, except that the former had less subendothelial deposits but focal onionskin changes in one intralobular artery in the transplant biopsy. A thrombus in an arteriole in one of renal transplant biopsies (Fig. 2C) further supports the thesis that the patient had recurrent thrombotic microangiopathy shortly after the renal transplantation. Although duplication of GBM is a key feature of chronic transplant glomerulopathy [7], the prominent GBM duplications within 40 days following renal transplantation favor a diagnosis of thrombotic microangiopathy rather than a chronic transplant glomerulopathy in this case. Thrombotic microangiopathy recurs in up to 41% of renal transplant cases and graft loss in the recurrent cases ensues in 70% of the cases [8,9]. Recurrent glomerulopathy has not been reported following Campath- 1H induction therapy [10-13]. In addition, there is a lack of endothelial staining of CD52 (the target of Campath-1H) in our experience (data not shown). We therefore believe that Campath-1H was not the factor that triggered the recurrent thrombotic microangiopathy. Acknowledgment The authors thank Dr. Helmut G. Rennke (Brigham and Women s Hospital, Boston, MA) and Dr. Michael Nalesnik (University of Pittsburgh Medical Center, Pittsburgh, PA) for consulting on Case 2. References 1. D Agati VD, Jennette JC, Silva FG. Non-neoplastic kidney diseases. In: Altas of Nontumor Pathology, First Series, Fascicle 4, Chapter 18, Armed Forces Institute of Pathology, Washington, DC, 2005; pp

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