—  SPECIALTY CONFERENCE  —

Renal Pathology

Case 1 - Acute Glomerulitis (Transplant Glomerulopathy) with Acute Tubular Injury and Vascular Immune Deposition Consistent with Antibody-mediated Acute Transplant Rejection

Cynthia C. Nast
Cedars-Sinai Medical Center
Los Angeles, CA





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Clinical History:

History of Present Illness: 53 year old Filipino female with end stage renal disease thought to be secondary to long standing severe hypertension. She underwent a deceased donor transplant (39 hours cold ischemia time) and induction therapy included steroids, thymoglobulin and mycophenolate. On POD #6 she was steroid-free, and tacrolimus was begun on POD#10, with subtherapeutic levels until POD#15 at which time the serum creatinine declined from a peak of 5.2 mg/dl to 1.8 mg/dl. Five days later the serum creatinine increased to 2.7 mg/dl at which time she also experienced hypotension and had a urinary tract infection for which IV vancomycin was administered.

Past Medical History: Severe hypertension as noted above with coronary artery disease. Remote history of tuberculosis.

Medications: mycophenolate, tacrolimus, septra nightly, cipro, vancomycin, valcyte, sporanox, magnesium oxide, labetolol, diltiazem, catapres, minoxidil, hydralazine,

Review of Systems: Noncontributory.

Physical Exam: BP: 130/63 T: 97.8 HR: 62 RR: 16
Chest: clear
Heart: regular rate, loud murmur
Abd: nontender, well-healed incision, no herniation.
Ext: 1-2+ bilateral lower extremity edema. Foley catheter draining clear urine
Neuro: Alert and oriented

Pathological/Microscopic Findings and any Immunohistochemical or Other Studies:
The light microscopy specimen contains five glomeruli which have many capillary luminal monocytes and lymphocytes with scattered neutrophils. Capillary walls segmentally are widened but with few double contours. The tubulo- interstitium, peritubular capillaries, arteries and arterioles lack inflammation. Tubular epithelial cells show acute injury with flattening and dilated lumina; few sloughed cells are in tubular lumina. Immunofluorescence microscopy shows fibrin (2-3+) in a linear pattern along glomerular capillary walls. Arterial and arteriolar walls stain for IgM (4+), C3 (3-4+), kappa (2+) and lambda (2+) in a granular to confluent granular pattern, and for fibrinogen (2-3+). C4d immunostain is negative in peritubular capillary walls. Electron microscopy reveals many monocytes with some lymphocytes and neutrophils within glomerular capillary lumina. Segmentally, there are moderately wide subendothelial lucent zones with flocculent material and early deposition of new subendothelial basement membrane material. Endothelial cells are swollen. Focally, peritubular capillary basement membranes have early multi- layering. Following the diagnosis of acute transplant rejection, likely antibody mediated and possibly cell mediated glomerular types, the patient was treated with steroids for five days. The serum creatinine improved from 2.7 to 1.5, but remained there. Repeat renal biopsy 12 days later revealed similar glomerular changes with slight reduction in inflammation, now with additional early collapsing features of focal and segmental glomerulosclerosis, and acute tubular necrosis with negative C4d staining. There was no inflammation in arteries, arterioles or peritubular capillaries. Glomerular capillary, artery and arteriolar walls stained for fibrin, with IgM and C3 staining of arteries and arterioles also (see images 12-15). Donor specific antibody testing results revealed: Solid-phase detecting IgG against single HLA antigens: negative for class I and II antibodies Endothelial cell crossmatch agains EC1 and EC2 by flow: negative Post-transplant crossmatch by flow: No pretreatement: T- and B-cell crossmatches positive Pronase pretreatment: T- and B-cell crossmatches negative Interpretation: Treatment with pronase rendered the T- and B-cell crossmatches negative. These results indicate the patient displays non-HLA antibodies which are not considered a contraindication to transplant. Over the next two weeks, the patient was treated with steroids, thymoglobulin, IVIG, and plasma exchange. The post-transplant crossmatch by flow was repeated at the end of this treatment with the same results. The creatinine initially dropped, and repeat biopsy at this time disclosed resolving glomerular inflammation and acute tubular injury (see images 16-17). The creatinine slowly rose over the ensuing 2-3 weeks, and the patient was treated with IVIG and plasma exchange. Repeat renal biopsy revealed further reduction in glomerular inflammation with continued acute tubular injury. C4d was negative in all repeat biopsies. The degree of IgM staining in artery walls was reduced, and one artery had fibin in the wall. At last follow-up, 11 months after the first renal biopsy, the patient is doing well with a serum creatinine level of 1.2 mg/dl.


Case 1 - Figure 1
The large artery wall is unremarkable. Tubular cells focally are flattened with dilated lumina but no inflammation.
Case 1 - Figure 2
The glomerulus is hypercellular but the tubulo-interstitium and artery have no inflammation.
Case 1 - Figure 3
Glomerular capillaries often are obliterated with inflammatory cells. There is no inflammation in the small artery or the walls of tubules.
Case 1 - Figure 4
Higher magnification of a glomerulus demonstrating many mononuclear leukocytes within capillary lumina. Capillary walls are predominantly single contoured.

Case 1 - Figure 5
There are lymphocytes, monocytes and few neutrophils in glomerular capillaries.
Case 1 - Figure 6
There is no tubulo-interstitial, peritubular capillary or vascular inflammation. Few tubular cells show loss of cytoplasmic volume with mild luminal dilatation.
Case 1 - Figure 7
Tubular epithelial cells are flattened and show acute injury.
Case 1 - Figure 8
Granular to confluent granular IgM within arterial walls.

Case 1 - Figure 9
Glomerular capillary walls with subendothelial flocculent material, swollen endothelium, and early deposition of new basement membrane material in the subendothelial area (double contour formation).
Case 1 - Figure 10
Multilayered peritubular capillary basement membrane.
Case 1 - Figure 11
C4d immunostain is negative.
Case 1 - Figure 12
Glomerulus with segmental podocyte hypertrophy,and cytoplasmic vacuoles and protein reabsorption droplets near the tubular pole.

Case 1 - Figure 12a
There is moderate glomerular inflammation primarily with mononuclear leukocytes. Note the arteriole has no abnormalities.
Case 1 - Figure 13
Thre is necrosis of tubular epithelial cells with a mitotic figure showing regeneration.
Case 1 - Figure 14
Arteriole with fibrin in the walls.
Case 1 - Figure 15
Glomerular capillary showing swollen endothelial cell cytoplasm, subendothelial lucency and new subendothelial basement membrane material.

Case 1 - Figure 16
Glomerulus with reduced inflammation and many patent capillary lumina.
Case 1 - Figure 17
Tubular epithelial cell acute injury.


Differential Diagnoses:
Cell mediated glomerular acute rejection Antibody mediated rejection Acute tubular necrosis

Final Diagnosis:
Acute glomerulitis (transplant glomerulopathy) with acute tubular injury and vascular immune deposition consistent with antibody-mediated acute transplant rejection.

Case Discussion:
Antibody mediated rejection (AMR) occurs in up to 50% of renal biopsies obtained for cause and is associated with acute and chronic allograft injury [1, 2, 3]. The morphologic features of early/active rejection include glomerular and peritubular capillary inflammation with neutrophils and monocytes, acute tubular injury and often glomerular capillary wall fibrin deposition with flocculent electron dense and lucent material in subendothelial zones. [2, 4] As the lesion progresses, glomerular capillaries display multilayered basement membranes and mesangiolysis may be evident (transplant glomerulopathy). Peritubular capillaries also develop multilayered basement membranes often in association with tubulo-interstitial scarring, loss of peritubular capillaries and possibly arterial neointimal thickening [5, 6]. Active AMR has been shown to associate closely with covalent binding of C4d to peritubular capillary walls where the C4d co-distributes with type IV collagen and is a marker for the complement activation associated with AMR. In addition, arterial and arteriolar wall IgG and/or IgM with C3 may be seen in AMR, often in association with fibrin in the walls and/or lumina. A definitive diagnosis of AMR requires morphologic and clinical components, including renal parenchymal injury with one or more of the features indicated above, positive C4d stain in peritubular capillaries, and the presence of donor specific antibodies (DSA) [7]. If two of these three components are present, a presumptive diagnosis of AMR often is made. C4d staining portends a worse prognosis for acute rejection, independent of other known predictors of rejection outcome [8, 9, 10]. However, the reliability of these criteria has been called into question based on several observations including the lack of C4d in some cases of transplant glomerulopathy, the lack of predictive value of anti-HLA antibodies for graft loss, and the persistence of DSA following clinically successful treatment of AMR [11, 12]. Investigators have reported poor graft survival associated with peritubular capillary and interstitial inflammation, but not with C4d positivity [13]. Additionally, patients with subclinical AMR who are C4d negative have been recognized recently and reported to have a long term prognosis intermediate between those with C4d+ AMR and without AMR [14]. Therefore, C4d staining is not an absolute marker for the presence or absence of AMR. AMR traditionally has been considered a consequence of antibodies directed against major MHC class I and class II antigens [15]. There also may be antibodies to other minor histocompatibility or non-classical MHC antigens such as MHC class I polypeptide-related sequences A (MICA) and B (MICB) which can cause acute rejection and graft loss [16, 17]. Binding of IgG or IgM anti-donor antibodies to the allograft endothelium with activation of complement is the predominant mechanism by which DSA exert their effect on the transplanted organ. These antibodies generally work through the classical pathway, activating C1 through interaction with the C1q globular domain with subsequent downstream activation of C2 and C4. Activation of C4 results in the C4d split product which then covalent binds to the peritubular capillaries as a marker of AMR. However, complement activation is not necessary for endothelial cell activation and proliferation in response to MHC class I antibodies [18, 19]. In particular, non-HLA antibodies and autoantibodies may induce allograft injury and can function in a non-complement fixing manner [20]. This has been demonstrated in a heart allograft model in vivo, in which antibody to class I MHC antigens induced chronic arterial lesions without complement participation [21]. Non-complement mechanisms may involve antibody cell lysis through an Fc receptor on the surface of natural killer cells and macrophages, or by inducing apoptosis of the target cell after binding through pathways such as mTOR [22, 23]. The diagnosis of AMR in the absence of C4d staining of peritubular capillaries may be difficult. C4d staining now is considered a hallmark of AMR; even in the setting of chronic AMR, C4d often remains positive [24]. However, the features of active AMR may be non-specific, making a diagnosis of AMR without C4d staining problematic. A recent study has suggested that C4d negative AMR is twice as common as C4d+ AMR and is a significant cause of late renal allograft loss [25]. Therefore, other methods for identification of AMR are necessary when C4d is negative, as morphologic findings may be insufficient even in the presence of detected DSA. One recent approach entails evaluating intragraft endothelial gene expression in kidney transplant biopsies [26]. This study by Sis et al identified increased endothelial associated transcripts in all forms of rejection, but with the highest expression in AMR with associated DSA. Notably, only 40% of kidneys with increased endothelial gene expression and chronic AMR or graft loss were positive for C4d. The diagnosis of AMR does require the presence of DSA; therefore, identification of classical as well as non-HLA antibodies and autoantibodies is necessary, with the understanding that non-complement fixing antibodies or antibodies against minor antigens may be clinically important, as in this case.

Conclusion(s):
AMR may occur in the absence of peritubular capillary C4d staining and be difficult to identify. A diagnosis of AMR in this setting requires recognition of the morphologic features of AMR such as glomerulitis, transplant glomerulopathy or peritubular capillaritis and correlation with DSA. However, these patients may have minor or non- classical DSA which should be sought and correlated with the morphologic and clinical findings. Newer methods of diagnosis, such as evaluation of endothelial gene expression, may be required to definitively identify patients with C4d negative AMR.

References:
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  2. Mauiyyedi S, Crespo M, Collins AB, et al. Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification. J Am Soc Nephrol 2002;13: 779–787.

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  7. Racusen LC, Colvin RB, Solez K, et al. Antibody-mediated rejection criteria: An addition to the Banff 97 classification of renal allograft rejection. Am J Transplant 2003;3:708–714.

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  17. Zou Y, Stastny P. The role of major histocompatibility complex class I chain-related gene A antibodies in organ transplantation. Curr Opin Organ Transplant. 2009 Aug;14 (4):414-8.

  18. Smith JD, Lawson C, Yacoub MH, Rose ML: Activation of NF-kappa B in human endothelial cells induced by monoclonal and allospecific HLA antibodies. Int Immunol 2000;12: 563– 571.

  19. Jin YP, Singh RP, Du ZY, Rajasekaran AK, Rozengurt E, Reed EF: Ligation of HLA class I molecules on endothelial cells induces phosphorylation of Src, paxillin, and focal adhesion kinase in an actin-dependent manner. J Immunol 2002;168: 5415–5423.

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  21. Hirohashi T, Uehara S, Chase CM, et al. Complement independent antibody-mediated endarteritis and transplant arteriopathy in mice. Am J Transplant 2010; 10: 510–517.

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