Section 3 -

Ultrastructural Evaluation of Renal Allograft Biopsies J. Allan Tucker University of South Alabama Mobile, AL USA

Introduction: Renal transplantation has become a common practice at many centers. Needle biopsies of renal allografts are frequently performed for allograft dysfunction and sometimes for surveillance. Table 1 lists some of the causes of renal allograft dysfunction to be considered in the evaluation of a renal allograft biopsy. Table 1 - Evaluation of Renal Transplant Biopsies Acute Tubular Necrosis Hyperacute Rejection Acute Antibody-Mediated Rejection Acute Cellular Rejection Chronic Rejection Drug Toxicity/Reaction Infection Hypertensive Renal Disease Miscellaneous Recurrent/De Novo Glomerular Disease Adapted from reference 1 Electron microscopy may, at times, contribute to the diagnosis in all of these areas. Electron microscopy particularly contributes to the evaluation of the following four categories: Chronic rejection Infection Miscellaneous Recurrent/de novo glomerular disease Chronic Rejection: Rejection is an important cause of renal allograft dysfunction and a common point of evaluation of renal allograft biopsies. Many centers apply the criteria of the Banff 97 working classification of renal allograft pathology [2]. By Banff 97 criteria, acute cellular rejection is recognized as a combination of interstitial inflammation and infiltration of the tubular epithelium by lymphocytes (tubulitis) and/or infiltration of the arterial intima by lymphocytes (intimal arteritis). The criteria for acute/active rejection are provided in table 2. Table 2 - Acute/Active Rejection [2] Type (Grade) Histopathological findings IA Cases with significant interstitial infiltration (>25% of parenchyma affected) and foci of moderate tubulitis (>4 mononuclear cells/tubular cross section or group of 10 tubular cells) IB Cases with significant interstitial infiltration (>25% of parenchyma affected) and foci of severe tubulitis (>10 mononuclear cells/tubular cross section or group of 10 tubular cells) IIA Cases with mild to moderate intimal arteritis (v1) IIB Cases with severe intimal arteritis comprising >25% of the luminal area (v2) III Cases with "transmural" arteritis and/or arterial fibrinoid change and necrosis of medial smooth muscle cells (v3 with accompanying lymphocytic inflammation) The Banff 97 classification also provides criteria for the evaluation of chronic/sclerosing allograft nephropathy, which is based on a combination of the extent of interstitial fibrosis and of tubular atrophy, as outlined in table 3 [2]. Table 3 - Chronic/Sclerosing Allograft Nephropathy [2] Grade Histopathological findings Grade I Mild interstitial fibrosis and tubular atrophy without (a) or (mild) with (b) specific changes suggesting chronic rejection Grade II Moderate interstitial fibrosis and tubular atrophy (a) or (b) (moderate) Grade III Severe interstitial fibrosis and tubular atrophy and tubular (severe) loss (a) or (b) Chronic rejection can result in these patterns of chronic/sclerosing allograft nephropathy, but a number of other disease states can produce similar changes, including renal ischemia, hypertension, drug effects, infection, increased ureteral pressure, and non-immune inflammatory processes [2]. Chronic/sclerosing allograft nephropathy, then, is a general term which does not specifically indicate chronic rejection. Each grade includes subdivision as (a) or (b) depending on whether specific changes suggesting chronic rejection are identified. By light microscopy, two major specific changes can be seen. The first is a characteristic arteriopathy consisting of arteriosclerotic-type thickening of the arterial intima, which is nonspecific, but which assumes a picture indicative of chronic rejection when mononuclear cells are present in the thickened intima. The other change which can be identified by light microscopy is the presence of chronic transplant glomerulopathy, with alterations in the glomeruli that include mesangial interposition which results in double contours of the basement membrane of peripheral capillary loops on PAS-stained sections. Electron microscopy can extend the ability to recognize chronic rejection in allografts with chronic/sclerosing allograft nephropathy, especially as the characteristic arteriopathy of chronic rejection is often not seen in affected allografts. Evaluation of the peritubular capillaries and the glomeruli ultrastructurally can reveal early evidence of chronic rejection that is not otherwise apparent. Peritubular capillaries normally exhibit a single, continuous basement membrane, and very slight lamellation of the membrane is commonly seen. Ultrastructural evaluation of allografts, however, may reveal extensive lamellation of the basement membrane of peritubular capillaries [3, 4]. Such lamellation can be seen in conditions other than chronic rejection and can sometimes be seen in native renal biopsies. Nonetheless, a very prominent degree of lamellation in the proper setting is highly indicative of chronic rejection. Ivanyi et al. concluded that when seven or more layers of basement membrane were identified, the finding was quite specific for chronic rejection [4]. Even if a portion of renal cortical tissue put aside for electron microscopy from an allograft biopsy does not yield any glomeruli, evaluation of the peritubular capillaries can be a useful and often the sole method of demonstrating specific changes of chronic rejection, changing a chronic/sclerosing allograft nephropathy grade from an "a" to a "b" subtype. While fully developed chronic transplant glomerulopathy can be identified by light microscopy. In such cases, electron microscopy reveals mesangial interposition and/or subendothelial lucency/flocculent material [3, 4]. Also, as with the peritubular capillaries, distinct lamellation of the glomerular basement membrane may be seen [5]. Glomeruli with early, mild chronic transplant glomerulopathy, however, often appear completely unremarkable by light microscopy. Some allograft biopsies with unremarkable-appearing glomeruli by light microscopy will reveal areas of early mesangial interposition, with mesangial cell cytoplasm extending from the paramesangium into a portion of the capillary loop, and may similarly reveal areas of mild subendothelial accumulation of lucent to flocculent material. Again, these early changes, undetectable by light microscopy, may be the only factor available to result in the change of the chronic/sclerosing allograft nephropathy grade from an "a" to a "b." Infection: Renal allografts are susceptible to infection, including viral infection. One common type of viral infection in these allografts is BK polyomavirus infection [6]. The renal tubular epithelial cells are particularly prone to this infection, and infected cells will sometimes display nuclear enlargement with smudging of the chromatin. Immunohistochemistry can confirm the presence of BK polyomavirus infection. Ultrastructurally, viral particles can be seen in the nucleus and also sometimes in the cytoplasm when the particles leak from the nucleus. These particles are generally round and crowded and measure about 40 to 45 nm [7]. One particularly useful morphologic feature for ultrastructural identification is that the particles will sometimes form crystalline arrays. Some laboratories uniformly perform immunohistochemical staining for this virus in allografts, and electron microscopy is often only confirmatory. Not all cases of infection, however, will produce the characteristic nuclear changes by light microscopy, and immunohistochemical stains, while sensitive, will not always yield optimal slides. Electron microscopy, then, may at times be a primary method of diagnosis. Further, electron microscopy may reveal other viral particles as well, such as cytomegalovirus [6, 7]. Miscellaneous: Ultrastructural examination may from time to time reveal any number of additional miscellaneous findings. For example, we have observed cases in which tubuloreticular inclusions were present in endothelial cells. These structures are often associated with systemic lupus erythematosus or HIV infection, though they are not specific [8]. While some authors report that tubuloreticular inclusions may be seen as a rare finding even in apparently normal kidneys [9], in our cases, the tubuloreticular inclusions were common and were seen in subsequent biopsies from the same patient as well. These patients did not have a positive ANA or a positive HIV test, and the significance of this finding remains to be seen, but such miscellaneous findings detected only with electron microscopy are no doubt of significance in some instances. Recurrent/De Novo Glomerular Disease: A renal allograft is subject to all of the same diseases as a native kidney. Further, for a patient undergoing transplantation for glomerulopathy, the original glomerular disease can sometimes recur in the allograft. Electron microscopy can be extremely useful in these cases. The following three cases are offered as an example of the importance of electron microscopy in the diagnosis of de novo glomerular disease. Case 1 History: A 53 year old woman who was 6 years status post renal transplantation for diabetes mellitus presented with a rising creatinine from 2.3 to 4.0. Light Microscopy: On routine sections, the glomeruli exhibited a possible slight increase in mesangial matrix but otherwise appeared unremarkable. On PAS-stained sections, a suggestion of an increased mesangial matrix was also seen, and, in addition, definite glomerular basement membrane duplication was seen. The findings, then, indicated chronic transplant glomerulopathy, as might be expected in this allograft. Electron Microscopy: Ultrastructural examination revealed very dramatic mesangial interposition. In addition, however, numerous subepithelial to intramembranous deposits were seen, some of which exhibited a microspherical substructure. The findings were conclusive for membranous glomerulopathy which was superimposed on chronic transplant glomerulopathy. Case 2 History: A 62 year old woman who was 5 months status post renal transplantation for adult polycystic kidney disease presented with a rise in creatinine and proteinuria. Light Microscopy: The glomeruli exhibited mild hypercellularity and an increase in mesangial matrix. PAS stain further demonstrated areas of apparent complexity of the glomerular basement membrane. The light microscopic findings, then, were suggestive of chronic transplant glomerulopathy, but the time course did not fit well for this entity. Electron Microscopy: By electron microscopy, mesangial interposition was indeed present, but in addition striking subendothelial and mesangial deposits were seen. Immunofluorescence staining was performed, revealing positive staining for IgG and C3. No tubuloreticular inclusions were seen, and the patient did not have a positive ANA. The findings, then, were diagnostic of de novo membranoproliferative glomerulonephritis. Case 3 History: The patient was a 49 year old man who was 15 years status post renal transplantation. The "underlying disease was unknown." The patient presented with a rise in creatinine and nephrotic range proteinuria (6 grams/24 hours). Light Microscopy: By light microscopy, the glomeruli appeared normocellular but exhibited some consolidation, probably from an increase in mesangial matrix, and some exhibited segmental to global glomerulosclerosis. Arteriolar thickening was noted. PAS-stained sections revealed similar findings as well as some possible basement membrane duplication. The findings, then, again indicated chronic transplant glomerulopathy and probably hypertension. Electron Microscopy: In the portion of tissue for electron microscopy, a glomerulus was not identified, but ultrastructural examination was performed. The tubules exhibited striking thickening of the tubular basement membrane, with areas measuring up to six microns in thickness. Though this finding is not specific, it raises the possibility of diabetes mellitus. Further, a minute portion of a somewhat crushed glomerulus was present at the edge of the tissue section which was not identified on the one micron sections. Examination of these capillary loops reveal no mesangial interposition, but marked thickening of the glomerular basement membrane was observed, with the thickness uniformly measuring greater than 1,000 nm. This finding indicated diabetes, which would also account for the arteriolar thickening. The clinician was contacted, and upon further review of the history, it was identified that the patient did have diabetes mellitus. The case, then, represented diabetic nephropathy occurring in an allograft. Conclusion: Because of the important contributions of electron microscopy in the evaluation of these allograft biopsies, electron microscopy is performed routinely on these samples at our institution. Electron microscopy proves particular useful in the following areas: Identification of specific features of chronic rejection Lamellation of basement membranes of peritubular capillaries Chronic transplant glomerulopathy Infection BK polyomavirus and other viral diseases Miscellaneous findings Tubuloreticular inclusions Recurrent/de novo glomerular disease References: D'Agati VD, Jennette JC, Silva FG. Pathology of renal transplantation. In: Non-Neoplastic Kidney Diseases. 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