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Peritubular capillaries in chronic renal allograft rejection: A quantitative ultrastructural study
Peritubular Capillaries in Chronic Renal Allograft Rejection: A Quantitative Ultrastructural Study ´ NYI, MD, HANAN FAHMY, MD, HOLLY BROWN, MD, BE´LA IVA ´ PAL SZENOHRADSZKY, MD, PHIL F. HALLORAN, MD, AND KIM SOLEZ, MD Peritubular capillaries (PCs) with a circumferentially multilayered basement membrane have been suggested as an ultrastructural indicator of chronic renal allograft rejection (CR). The authors validated this lesion as a marker for CR, by analyzing its quantitative features, specificity, and sensitivity in 169 renal biopsy specimens. The mean number of circumferential layers (PCcirc) and the incidences of the grades (mild: 2 to 4, moderate: 5 to 6, severe: 7 or more layers) were investigated in biopsy specimens involving CR (CRBx, n ⴝ 46), acute rejection (n ⴝ 11), normal kidneys (n ⴝ 20), psoriatics treated with cyclosporine (n ⴝ 13), renal transplants with chronic cyclosporine toxicity (n ⴝ 12), native kidney diseases (NKD, n ⴝ 56), and transplant nephrectomies attributable to CR (Crnephr, n ⴝ 11). CR was diagnosed with regard to the clinical features and the presence of intimal fibrosis in 41 biopsy specimens or transplant glomerulopathy in 35 biopsy specimens (cg; identified only by electron microscopy in 10 cases). NKD included chronic glomerulonephritis, chronic tubulointerstitial nephritis, benign nephrosclerosis, thrombotic microangiopathy, diabetic nephropathy, and renal disease in elderly patients (median age, 72 years). All PCs around glomeruli were sampled (median, 14 profiles per case). PCs with a moderate/ severe lesion appeared as serrated profiles with a thick, ribbon-like basement membrane layer in semithin plastic sections. The numbers of circumferentially multilayered PCs were significantly characteristic of CR (PCcirc in CRBx: 2.87 ⴞ 1.83 SD; range, 0 to 7.36; P < .001 v other groups). A severe lesion occurred exclusively in CR (in 12% of
the PCs in CRBx, and in 38% in CRnephr). A moderate lesion was observed in 0.6% of the PCs in NKD, 16% in CRBx, and 21% in CRnephr. Three or more PCs with a moderate lesion were encountered only in CR. A mild lesion was not suggestive of CR at all. In CRBx, 27 cases showed a severe lesion or 3 or more PCs with a moderate lesion (cpc; sensitivity: 59%). Four of the 27 cases lacked cg. The cumulative incidence of cpc and cg was 85%. In transplants with cyclosporine toxicity, the presence of cpc verified the coexistence of CR in 7 specimens. In conclusion, cpc is a specific marker of CR. The incidence of cpc increases as CR progresses. The lesion may be caused by a low-grade rejection injury to the PCs. Careful analysis of semithin sections promotes the better sampling of cpc. An ultrastructural demonstration of cpc and cg defines CR more precisely than does light microscopic evaluation per se. HUM PATHOL 31: 1129-1138. Copyright © 2000 by W.B. Saunders Company Key words: basement membrane, electron microscopy, native kidney disease, peritubular capillary, rejection, chronic allograft nephropathy. Abbreviations: CR, chronic renal allograft rejection; PC, peritubular capillary; PCcirc, mean number of circumferential basement membrane layers; cg, transplant glomerulopathy; cpc, a peritubular capillary with 7 or more circumferential basement membrane layers or at least 3 peritubular capillaries with 5 to 6 circumferential basement membrane layers.
Chronic allograft nephropathy is the major cause of renal allograft loss after the first year of transplantation. This nephropathy is a result of multiple injury/ stress factors. The immune factors involve panel-reactive antibodies, HLA match, immunosuppression, acute rejection, and drug compliance. The nonimmune factors include advanced donor age, ischemia-reperfusion injury, size mismatch, hypertension, cyclosporine trough levels, lipid disorders, recurrent disease, and so forth.1-6 The pathology of chronic allograft nephropa-
thy is nonspecific: interstitial fibrosis, tubular atrophy, unspecific arteriolopathy, unspecific glomerular changes, interstitial inflammation, lack of tubulitis, and HLA-DR–negative tubules are observed in the biopsy specimen.6 With the use of clinical and light microscopic criteria, a significant number of chronic allograft nephropathy cases can be classified into the narrower category of chronic renal allograft rejection (CR). Mihatsch et al6 recently reviewed 1,500 biopsies performed more than 6 months after transplantation. Thirty-five percent of the cases were diagnosed as chronic transplant nephropathy, 14% as CR, and 6% as acute and CR.6 The relatively low percentage of cases of CR raises the question of whether morphologic alterations caused by immunologic damage to the graft have become infrequent in the cyclosporine era, or whether standard light microscopy per se is insufficient for the diagnosis of CR in a certain number of cases involving CR. The aim of this article is to show that peritubular capillaries (PCs) undergo ultrastructural changes specific for CR (the cpc lesion), and that a search for the cpc lesion facilitates the diagnostic accuracy of CR. CR is a clinicopathologic entity6,7 characterized by a progressive decline in transplant function more than 3 months posttransplantation, with a compatible histology, and is not attributable to other known factors such
From the Department of Pathology, Albert Szent-Gyo¨rgyi Medical University, Szeged, Hungary; the Department of Laboratory Medicine and Pathology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada; the Department of Pathology, University of Calgary, Calgary, Alberta, Canada; the Renal Transplantation Unit, Clinics of Surgery, Albert Szent-Gyo¨rgyi Medical University, Szeged, Hungary; and the Division of Nephrology and Immunology, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada. Accepted for publication June 13, 2000. Supported in part by grants to Be´la Iva´nyi (OTKA T-025590, and ETT-588/1996-06, Budapest, Hungary). Address correspondence and reprint requests to Be´la Iva´nyi, Department of Pathology, Albert Szent-Gyo¨rgyi Medical University, Kossuth L. sgt. 40, H-6724 Szeged, Hungary. Copyright © 2000 by W.B. Saunders Company 0046-8177/00/3109-0022$10.00/0 doi: 10.1053/hupa.2000.16677
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as cyclosporine toxicity, obstructive uropathy, glomerulonephritis, hypertensive vascular disease, or diabetic nephropathy. The cardinal histopathologic features of CR are intimal fibrous thickening in arteries, and transplant glomerulopathy (cg).6,8 The diagnostic value of these features, however, is influenced by the fact that intimal fibrosis is not entirely specific (it also develops in aging, hypertension, etc.), and the incidence of cg varies in different series. Markers have therefore been sought that would enhance the accuracy of CR diagnosis. In 1990, Monga et al9 reported that in CR the basement membrane of the peritubular capillaries (PCs) undergoes circumferential multilayering and splitting, and the lesion was later proposed as an ultrastructural indicator of CR.10-15 However, unresolved questions remain as concerns the interpretation of the lesion. The criteria of focal or circumferential multiplication and the grades of circumferential multilayering were not precisely defined in the initial publications,9-13 and validation of the specificity of the lesion still requires the study of appropriate controls. Furthermore, the diagnostic sensitivity of the lesion is not known sufficiently accurately. To answer these questions, we performed an ultrastructural analysis of PCs in 169 renal biopsy specimens. Three parameters, the mean number of circumferential layers, the grade of circumferential multilayering, and the incidences of the grades, were used for a quantitative characterization of the state of the PC basement membrane in biopsy specimens involving CR and in controls. The latter group consisted of selected series of native kidney diseases, psoriatics treated long-term with cyclosporine, normal healthy individuals, acute rejection, and transplant nephrectomies caused by CR. The native kidney diseases included chronic glomerulonephritis, chronic tubulointerstitial nephritis, hypertensive kidney disease, scleroderma kidney, thrombotic microangiopathy, diabetic glomerulosclerosis, and elderly patients with renal parenchymal disease. Cases were chosen in which the clinical features or light microscopic lesions resembled the situation in CR. Because double-contoured glomerular capillary walls are a common feature of thrombotic microangiopathy and cg, we were interested in whether the basement membrane of the PCs in thrombotic microangiopathy undergoes circumferential multilayering. Aging and cyclosporine administration might influence the grade of basement membrane multilayering of the PCs. We therefore investigated the basement membrane of PCs in elderly patients and in psoriatics treated with cyclosporine. Once the diagnostic experience with the circumferential PC lesion had been acquired, PCs in transplant biopsies involving chronic cyclosporine toxicity also were studied for the possible presence of coexisting CR. MATERIALS AND METHODS Originally, the biopsy specimens were investigated by light microscopy, direct immunofluorescence (IgG, IgA, IgM, C3, C1q, and fibrinogen), and electron microscopy, accord-
ing to standard methods. The specimens from psoriatic patients were studied by light and electron microscopy. Electron microscopy was used to diagnose cg in cases with a Banff score of cg0 (double contours in ⬍10% of peripheral capillary loops in most severely affected glomerulus).8 The ultrastructural criteria of cg were at least a twofold thickening of the capillary wall as a result of the expansion of the subendothelial space by amorphous or lace-like material and the formation of new basal lamina with or without mesangial cell interposition in at least 3 capillary loops. If electron microscopy confirmed the presence of cg, the Banff score was corrected to cg0.5. At least 1 glomerulus per case was examined.
Quantitative Analysis of Circumferential Basement Membrane Multiplication The thin sections were placed on large slotted grids (2 ⫻ 1 mm), and all PC profiles around glomeruli were sampled and evaluated by one of us (B.I.) without knowledge of the Banff chronicity scores or group assignments (median, 14 profiles per case; range, 8 to 31). The capillary circumference was divided into 4 portions by the longest axis and the longest axis perpendicular to this. The multilayering of the basement membrane was regarded as circumferential if it was seen in at least 3 of the 4 portions of the capillary circumference. The site of reading of the basement membrane layers was the narrow peritubular interstitium. The change was recorded as PCs with 2 layers (Q2), with 3 or 4 layers (Q3-4), with 5 or 6 layers (Q5-6), with 7 or 8 layers (Q7-8), or with more than 8 layers (Q9). The mean number of circumferential layers (PCcirc) was calculated by dividing the total number of circumferentially layered PC profiles by the total number of PC profiles (Qtot): PC circ ⫽ 关共Q 2 ⫻ 2兲 ⫹ 共Q 3-4 ⫻ 4兲 ⫹ 共Q 5-6 ⫻ 6兲 ⫹ 共Q 7-8 ⫻ 8兲 ⫹ 共Q 9 ⫻ 9兲兴/Q tot The grade of circumferential multilayering was arbitrarily defined as mild (2 to 4 layers), moderate (5 to 6 layers), or severe (ⱖ7 layers). The focal multilayering of the PC basement membrane was regarded as abnormal if at least 3 layers were observed.
Study Groups Chronic Rejection, Biopsy Group. The biopsies were performed between 1991 and 1999 (Table 1). The biopsy specimens had to meet criteria of specimen adequacy (10 or more glomeruli with at least 2 arteries). Cases of CR were selected on the presence of fibrous intimal thickening in arteries or cg, the absence of severe hyaline arteriolopathy indicative of chronic cyclosporine nephrotoxicity, an insidious increase in the serum creatinine level, no history of infection, obstructive uropathy, recurrent glomerulonephritis, diabetes, or severe hypertension (ⱖ180/110 mm Hg). The cases met the definition of being characterized by a progressive decline in transplant function occurring more than 3 months after transplantation in the absence of any other known disease. The cases from Edmonton were chosen at random, and the cases from Szeged were consecutive. The study group comprised 46 biopsy specimens from 35 Hungarian and 11 Canadian patients. Forty-four patients received a cadaveric kidney, and 2 patients received a living related donor kidney. The patients were immunosuppressed with cyclosporine and prednisolone, or cyclosporine, prednisolone, and azathioprine. The dose of cyclosporine was 3 to 5 mg/kg/d, adjusted individually according to the serum cyclosporine level, posttransplantation period, and so forth. Before biopsy, 12 patients
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TABLE 1. Clinical Data in Chronic Rejection and Native Kidney Diseases (Median, Range)
Chronic rejection, biopsy group Chronic rejection, nephrectomy group Chronic sclerosing glomerulonephritis Chronic tubulointerstitial nephritis Hypertensive kidney disease Thrombotic microangiopathy/ scleroderma kidney Diabetic nodular glomerulosclerosis Elderly patients with renal disease
Male, Female
Age (yr)
BP, Systolic (mm Hg)
BP, Diastolic (mm Hg)
Serum Creatinine (mg/dL)
Posttransplantation Period (mo)
24, 22
43, 12-58
140, 100-180
90, 70-110
3.86, 1.79-12.5
21, 3-240
5, 6
44, 38-55
150, 85-175
95, 85-115
on dialysis
41, 9-156
6, 3
45, 35-60
170, 150-190
105, 80-140
4.07, 1.71-10.55
1, 7 5, 2
38, 18-46 52, 18-65
158, 120-205 180, 150-230
100, 80-125 105, 86-130
4.38, 1.68-11.36 4.54, 2.55-7.68
2, 6
48, 7-77
174, 155-200
100, 80-110
5.96, 0.93-13.63
4, 6
45, 24-71
170, 130-190
90, 80-110
1.68, 0.95-4.15
10, 4
72, 67-82
155, 110-210
87.5, 80-110
1.81, 0.98-5.98
Abbreviations: BP, blood pressure.
had mild to moderate hypertension. Thirty-two grafts terminated in chronic renal failure necessitating hemodialysis (median postbiopsy period: 7 months; range, 0 to 20). Twelve grafts are still functioning, with a serum creatinine level below 4 mg/dL in 6 patients. One patient died 10 months after biopsy (serum creatinine value at death: 2.72 mg/dL). Follow-up data were not available in 1 patient. Chronic Rejection, Nephrectomy Group. Intraoperative wedge biopsy specimens were studied from 11 Hungarian patients, all receiving cyclosporine and steroids, who underwent transplant nephrectomy because of CR (Table 1). The patients received transplanted cadaveric kidneys. Native Kidney Diseases. Forty-seven patients were from Hungary, and 9 patients were from Canada. In the group of chronic sclerosing glomerulonephritis, the patients showed an insidiously developed deterioration of the renal function. Four patients had marked proteinuria, with the nephrotic syndrome in 2 patients. In chronic nonbacterial tubulointerstitial nephritis, 2 cases were associated with Sjo¨gren’s syndrome, and 1 case with anterior uveitis. Hypertensive kidney disease was diagnosed by using the following criteria: a history of elevated blood pressure, normally positioned kidneys without scarring or signs of obstructive nephropathy, a negative search for a cause of secondary hypertension, normal blood glucose levels, and the morphologic demonstration of hyaline arteriolosclerosis and the absence of glomerular disease (Table 1). Morphologically, all cases with glomerulonephritis, tubulointerstitial nephritis, or hypertensive kidney disease showed marked interstitial fibrosis and tubular atrophy. Thrombotic microangiopathy occurred in 6 patients and was characterized by acute renal failure, anemia, and thrombocytopenia, with hemolysis in 4 patients, and prodromal diarrhea in 2 patients. Scleroderma kidney was observed in 2 patients; it displayed intimal thickening in arterioles and interlobular arteries, partial glomerular sclerosis, and a prominent juxtaglomerular apparatus. In diabetic nodular glomerulosclerosis, 6 patients had type 1 diabetes, and 4 patients had type 2 diabetes. The biopsy diagnoses in elderly patients were renal amyloidosis: 2 cases; primary glomerular disease: 7 cases; antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis: 3 cases; and hypertensive kidney disease: 2 cases. Normal Kidneys. The group comprised biopsy specimens from 5 Danish psoriatic patients with a normal kidney function, who underwent biopsies before long-term treatment with cyclosporine, and 15 baseline biopsy specimens from 8
living related donors and 7 cadaver donors. The baseline biopsy specimens, all from Edmonton, were obtained 1 hour after the completion of vascular anastomoses. Cyclosporine-Treated Native Kidneys. During the past decade, a considerable number of adult Danish psoriatics have been treated with long-term low-dose cyclosporine (2.5 to 6 mg/kg/d). The patients were rigorously controlled and regularly underwent biopsies to diagnose cyclosporine nephrotoxicity.16 Thirteen biopsy specimens, including 3 rebiopsies, were originally embedded for electron microscopy, and these were analyzed in the current study. The biopsy specimens were from 6 men and 4 women, treated for a median of 3 years (range, 1 to 5). At biopsy, the patients had normal blood pressure and blood sugar levels, and the median serum creatinine value was 1.06 mg/dL (range, 0.67 to 1.38). The light microscopic severity of chronic cyclosporine toxicity was assessed semiquantitatively by B.I. The degrees of arteriolopathy, interstitial fibrosis, tubular atrophy, hypertrophy of juxtaglomerular apparatuses, totally sclerosed glomeruli, and glomeruli with segmental lesions were scored. The sum of the scores expressed the severity of nephrotoxicity in the individual cases (median severity, 6; range, 2 to 9). Acute Rejection. The patients were from Canada (9 men and 2 women; median age, 39 years; range, 18 to 63) and were selected according to their Banff scores and typical clinical presentation. All were taking cyclosporine and had a good initial renal function, followed by an increase of at least 50% in the serum creatinine level versus the baseline value. After biopsy, all responded to anti-rejection therapy with a decline of at least 30% in the serum creatinine level. The median serum creatinine value at biopsy was 2.67 mg/dL (range, 1.93 to 6.48). The biopsies were performed a median of 39 days after transplantation (range, 10 to 252). The Banff acute scores8 were: v1, 9; v2, 2; g0, 10; g1, 1; i2, 7; i3, 4; t1, 2; t2, 7; and t3, 2. Chronic Cyclosporine Toxicity in Renal Transplants. PCs of 12 consecutive biopsy specimens from cadaveric transplant Hungarian patients with chronic cyclosporine toxicity (9 men and 3 women; median age, 42.5 years; range, 25 to 61) were evaluated. Cyclosporine toxicity was diagnosed on the basis of the presence of severe hyaline arteriolopathy in the biopsy specimen, an insidiously increasing serum creatinine level, and no history of diabetes, infection, recurrent glomerulonephritis, or severe hypertension. The median serum creatinine value at biopsy was 3.55 mg/dL (range, 2.5 to 5.34), and the
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median period between transplantation and biopsy was 50.5 months (range, 7 to 107). Four patients had mild hypertension. After the omission of cyclosporine, 6 patients exhibited a decrease in the serum creatinine level. The median graft survival in 5 patients was 20 months (range, 10 to 32). Six patients currently have functioning grafts with an acceptable function after a median follow-up period of 6 months (range, 4 to 10). One patient died 15 months after biopsy; his graft function was moderately impaired. The vascular and glomerular Banff chronicity scores8 were cv0, 1; cv1, 8; cv2, 3; cg0, 6; cg0.5, 3; cg2, 1; cg3, 2; ah3, 12.
Statistics Differences between the study groups were analyzed by the Wilcoxon test. Statistical significance was accepted at a probability level of 5%. The Banff chronicity scores and the PCcirc values in the biopsy group of CR were correlated by the Kendall test. The level of significance was 1%.
RESULTS Features of Biopsy Group of CR The clinical and morphologic data from the Edmonton and Szeged series are compared first. The 2 series then are combined, and the overall data are given. To facilitate reading, only relevant data are mentioned numerically. There was no significant difference between the series from Edmonton and that from Szeged in terms of posttransplantation period, or cv and cg scores. Significantly lower values were found in the Edmonton series as regards the ci and ct scores, PCcirc values, and serum creatinine levels (median value for Edmonton, 2.8 mg/dL; and for Szeged, 3.76 mg/ dL). In the combined material, the vascular and glomerular Banff chronicity scores were cv0, 5; cv1, 13; cv2, 18; cv3, 10; cg0, 11; cg0.5, 10; cg1, 15; cg2, 9; cg3, 1; ah0, 15; ah1, 18; and ah2, 13. The Banff acute scores showed superimposed acute rejection in 36 patients (acute tubulointerstitial rejection, 30; acute vascular and tubulointerstitial rejection, 6) and the suspicion of acute rejection in 8 patients. Morphology of Circumferential Basement Membrane Multilayering Normally, PCs in the cortical labyrinth consists of an extremely flat, fenestrated endothelium that rests on a thin basement membrane (Fig 1). The
severe (Figs 2 and 3) or moderate lesions (Fig 4) had tightly packed basement membrane layers, which were frequently split and usually were devoid of collagen fibers. The endothelium was nonfenestrated and had a serrated contour along the interstitial aspect of the cell body. The mild lesion contained collagen fibers within the loosely arranged basement membrane layers (Fig 5), splitting was infrequent, and the endothelium was fenestrated rather than nonfenestrated. The number of layers was usually higher along the wide interstitial aspect than along the narrow interstitial aspect of the PC. On occasion, the layers fused into a thickened basal lamina. Irrespective of the grade of the lesion, the capillary wall sometimes contained lymphocytes (observed in 16 cases). Interstitial myofibroblasts were frequently localized around the PCs. In 10 microvessel profiles from 8 specimens, an ultrastructural distinction between an efferent arteriole and a PC with a myofibroblast layer was impossible, and these profiles were excluded from further analysis. In semithin plastic sections, PCs with a moderate/severe lesion appeared as serrated profiles with a thick, ribbon-like basement membrane layer (Fig 6; compare with normal PCs in Fig 7). PCcirc Values and Incidences of Grades of PCs with Circumferentially Multilayered Basement Membrane In the biopsy group of CR, a severe lesion was recorded in 21 specimens. It was present in 3 specimens that did not display cg. PCs with a moderate lesion but without a severe lesion were noted in 16 grafts. Among these, 6 grafts had 3 or more PCs with a moderate lesion. Of the 6 grafts with 3 or more PCs with a moderate lesion, 1 graft lacked cg. A mild lesion or PCs without a circumferential multilayered basement membrane were recorded in 9 grafts. Moderate and severe multilayering occurred more frequently and extensively in the graftectomies than in the biopsy group of CR (Figs 8, 9), with correspondingly higher PCcirc values in the graftectomies (P ⬍ .01). A severe lesion and 3 or more PCs with a moderate lesion occurred exclusively in CR (the cpc lesion). Twenty-seven patients had cpc lesion (sensitivity: 59%), whereas 35 patients had cg 0.5 or greater (sensitivity, 76%). The cumulative incidence of cpc and cg was 85%. A mild lesion was not suggestive
FIGURE 1. A normal peritubular capillary from the cortical labirynth: The flat and fenestrated endothelium rests on a thin basement membrane. IS, narrow peritubular interstitium; lumen, lumen of the capillary; TBM, tubular basement membrane. (original magnification ⫻ 21,000.)
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FIGURE 2. Severe lesion: Circumferential multiplication and splitting of the basement membrane along the wide and narrow peritubular interstitium. The capillary has a serrated contour, the endothelium (END) is thickened and nonfenestrated, and the number of basement membrane layers is 7. IS, narrow peritubular interstitium; lumen, lumen of the capillary. (original magnification ⫻ 22,000.)
of CR at all. There was no significant correlation between the Banff chronicity scores and the PCcirc values (Table 2; P ⬎ .5 for all scores). In native kidney diseases, a moderate lesion was recorded in 2 PCs in 1 case of chronic tubulointerstitial nephritis (PCcirc value, 2.63). This case was not associated with the Sjo¨gren syndrome. In 2 cases of thrombotic microangiopathy (PCcirc values, 2 and 2.78), 1 and 2 PCs, respectively, displayed a moderate lesion.
Renal Transplants With Chronic Cyclosporine Toxicity Cyclosporine toxicity and CR coexisted in 7 biopsy specimens, confirmed by the presence of cpc in 7 specimens. Six of the 7 specimens also displayed cg, which was identified only ultrastructurally on 3 occasions. The vascular scores were cv0, 1; cv1, 4; and cv2, 2. In the remaining 5 cases (cv1, 4 and cv0, 1), the
FIGURE 3. Severe lesion: multiplication (at least 10 layers) and splitting of the basement membrane. Collagen fibers are absent in the area of basement membrane change. The endothelium (END) is thickened and nonfenestrated, and the lumen contains 3 lymphocytes. IS, narrow peritubular interstitium. (original magnification ⫻ 31,000.)
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FIGURE 4. Moderate lesion: Five to six circumferential layers of the basement membrane, devoid of collagen. IS, narrow peritubular interstitium; lumen, lumen of the capillary. (original magnification ⫻ 27,000.)
ultrastructural search for cpc and cg provided negative results. DISCUSSION Previous publications on circumferential multilayering of the PC basement membrane did not describe the quantitative changes adequately. This is because the PCs are understudied in renal pathology, and the methodology for assessing quantitative PC changes is undergoing development. Our study provides clues for a quantitative analysis of PCs in CR. We sampled all available PC profiles, the sampling being performed exclusively from the cortical labyrinth. It was important to exclude terminal profiles of arterioles, because the basal lamina of the arterioles in cyclosporine-treated patients shows multilayering,17 a change similar to that seen in PCs in CR. In our material, differentiation of a myofibroblast-encircled PC profile from an efferent arteriolar profile was impossible on only 10 occasions, a negligible proportion in the determination of the incidences of circumferentially multilayered PCs. The circumferential lesion was defined as seen in at least 75% of the capillary perimeter. Drachenberg et al14 defined the basement membrane multilayering as circumferential if it was seen in more than 60% of the vessel’s circumference. The stricter criterion that we used re-
duced the possibility of interpreting a focal lesion as circumferential. In a previous ultrastructural stereologic study, PCs from the cortical labyrinth were examined in renal graft biopsy specimens involving acute rejection.18 Hypertrophy of endothelial cells, a disappearance of fenestration, an increased adherence and passage of lymphocytes, and defects in the endothelial lining were demonstrated. The observations showed that during acute rejection PCs undergo a postcapillary venule-like transformation that enhances the influx of inflammatory cells into the graft. Because CR and subclinical acute rejection may coexist in biopsy specimens at 3 months or more after transplantation,19 in the current study, we decided to analyze the multiplication of the basement membrane of PCs that seemed to reflect chronic damage and was easy to read, and not to deal with endothelial cell changes that may be influenced by the acute rejection process. Reproducibility studies on the evaluation of the PC basement membrane lamination have not been performed yet. In our experience, PCs with a severe or moderate lesion cannot be misinterpreted. The serrated outline of the endothelial cells and splitting of the basement membrane, however, may be absent in PC profiles that are transitional between the mild and moderate grades. In such a situation, the evaluation must be strictly confined to the narrow peri-
FIGURE 5. Mild lesion: Collagen fibers within the 2 to 3, loosely arranged layers of the basement membrane. IS, narrow peritubular interstitium; TBM, tubular basement membrane; lumen, lumen of the capillary. (original magnification ⫻ 25,000.)
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FIGURE 6. Moderate/severe peritubular capillary lesions in semithin plastic section: The 2 capillaries (PC) have a ribbonlike basement membrane, and serrated contour. (Toluidine blue, original magnification ⫻1,000.)
tubular interstitium. If there is any difficulty in the determination of the number of layers (focal fusion of the layers, the narrow and wide interstitium cannot be recognized properly, or more layers are present on the opposite side of the narrow peritubular interstitium), the lesion should be categorized into the lower grade. If future investigations on the PCs in CR adopt this mode of evaluation, the chance of misinterpretation of the circumferential PC lesion seems low. Although fibrous intimal thickening is a strong indicator of CR, the diagnostic specificity of the lesion is reduced if the recipient has hypertension or diabetes, or the recipient received a graft from a hypertensive or aged donor. To assess whether the multilayering of the PC basement membrane is independent of the conditions listed above, for the first time in the literature we applied highly selected series of controls. In the study by Drachenberg et al,14 85 consecutive biopsy specimens with native kidney disease and 6 allografts with diseases other than cg were evaluated as controls. Their definitions of the circumferential lesion and the severe grade were similar to ours. This fortunate situation allows a combination of the results of the 2 studies in terms of the specificity of circumferential multilayering. Accordingly, the overall controls comprise 174 native kidney and 17 allograft biopsy specimens. The most important conditions include normal kidney, aging kidney, chronic cyclosporine nephrotoxicity in native kidney, hypertensive kidney disease, primary sclerosing and nonsclerosing glomerular diseases, diabetic nephropathy, amyloidosis, acute and chronic tubulointerstitial nephritis, and acute rejection. In both studies, the severe lesion was encountered only in CR, but not in the controls. A moderate PC lesion was a characteristic feature of CR in our material. However, the lesion occurred in 2 cases of thrombotic microangiopathy and 1 case of chronic tubulointerstitial nephritis in our series, and in 2 cases of thrombotic microangiopathy in the Drachenberg series. These findings indicate that thrombotic microangiopathy can be associated with a moderate degree of circumferential PC membrane multilayering. Although not documented appropriately in the literature, obstructive uropathy, analgesic nephropathy and radiation nephritis potentially might
show multilayering of the PC basement membrane in the cortical labyrinth. We recently examined the PCs in a native end-stage kidney from an adult patient with reflux nephropathy. Of the 9 periglomerular PCs studied, 1 displayed a severe lesion, 5 a moderate lesion, and 3 a mild lesion. This finding draws attention to the need to collect cases with obstructive uropathy before the end-stage phase to determine whether the condition is associated with severe circumferential multilayering of the PC basement membrane. We conclude that if obstructive uropathy, thrombotic microangiopathy, analgesic nephropathy, and radiation nephritis are excluded clinically in a graft with a late dysfunction, cpc can be used as a specific marker for CR. The sensitivity of cpc was 59%. The incidence of a severe PC lesion in the Drachenberg series was 57%.14 These figures show that PC lesions specific for CR occur in approximately 60% of the cases with CR. A 60% sensitivity may seem low as a reliable marker of CR, but it must not be forgotten that cpc can be used safely for the diagnosis of CR in those biopsy specimens in which the specificity of arterial intimal fibrosis may be questioned, or cg is lacking (we had 4 such cases). The diagnostic value of cpc was justified in our transplant patients with chronic cyclosporine toxicity, in whom the presence of cpc verified the coexistence of CR in more than half of the cases. Earlier studies did not specify how to recognize the circumferential multilayering of the PC basement membrane light microscopically. On reviewing the semithin sections, we found that the severe/moderate PC lesions had a typical appearance: a serrated outline of the endothelial cell body along the interstitial aspect of the PC, underlined by thickened, ribbon-like basement membrane material. A careful analysis of semithin sections, therefore, may promote a better sampling of cpc. The serrated contour of the PCs may be caused by the shrinkage of the microvessel. The pathogenesis of the circumferential lamination and splitting of the basement membrane of PCs is not known precisely. It may be recalled that, by express-
FIGURE 7. Normal peritubular capillaries (asterisks) in semithin plastic section from a patient with chronic rejection. (Toluidine blue, original magnification ⫻1,000.)
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FIGURE 8. Peritubular capillaries with circumferentially multilayered basement membrane occurred frequently in chronic rejection. The incidence increased, as chronic rejection progressed. Abbreviations: Normal, healthy individuals; CyA, psoriatics treated with cyclosporine; AR, acute rejection; NKD, native kidney diseases; CRBx, biopsy group of chronic rejection; Crnephr, nephrectomy group of chronic rejection.
ing HLA class I and class II molecules, PCs are the targets of rejection responses.20-22 The transcapillary passage of inflammatory cells has been shown to be associated with mild defects in the microvessel wall.23 Because circumferentially multilayered PCs were significantly characteristic of CR (P ⬍ .001 v other groups), it is likely that the development of cpc is related to rejection injury to the PCs. Our data showed that circumferentially multilayered PCs were focally distributed in the cortical labyrinth and, as CR progressed, the distribution of PCs showing a circumferential lesion broadened, and the grade of the lesion worsened. The focal nature of cpc does not favor antibody-mediated rejection mechanisms. In the current study, lymphocytes in the PC wall were noted in 16 specimens elec-
tron microscopically, and light microscopic evidence of acute tubulointerstitial rejection was recorded in 78% of the cases. Protocol biopsy specimens from patients with a clinically stable graft function showed that subclinical rejection of the graft had occurred in a significant number of cases,5,19 and untreated subclinical rejection was associated with a late graft dysfunction.24 The high frequency of concomitant acute rejection is suggestive of the idea that the cpc lesion is attributable to repeated or ongoing slowly progressive, smoldering cell-mediated rejection injury to the PCs. The target antigens may be either the classical HLA antigens or cryptic antigens exposed during previous tissue damage.3 The PCcirc values did not correlate with the grades
FIGURE 9. The severe lesion was specific, and the moderate lesion was highly suggestive for chronic rejection. The mild lesion was not indicative for chronic rejection. Abbreviations: Normal, healthy individuals; CyA, psoriatics treated with cyclosporine; AR, acute rejection; NKD, native kidney diseases; CRBx, biopsy group of chronic rejection; Crnephr, nephrectomy group of chronic rejection.
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TABLE 2.
Mean Number of Circumferential Basement Membrane Layers
Normal Cyclosporine-treated psoriatics Acute rejection Native kidney diseases Chronic rejection, biopsy Chronic rejection, nephrectomy
PCcirc ⫾ SD
Range of PCcirc
0.02 ⫾ 0.06 0.03 ⫾ 0.14 0.26 ⫾ 0.3 0.53 ⫾ 0.65 2.87 ⫾ 1.83* 5.48 ⫾ 2.02
0-0.21 0-0.5 0-0.89 0-2.78 0-7.36 2.28-8.14
Abbreviation: PCcirc, mean number of circumferential basement membrane layers. * Significantly different from nephrectomies (P ⬍ .01) and other groups (P ⬍ .001). The difference between native kidney diseases vs acute rejection was also significant (P ⬍ .02).
of cg. This finding was surprising, because cpc and cg coexisted in most of the CR cases. The lack of a mathematical correlation, however, does not automatically exclude a biologic correlation. The reasons for no correlation may include the wide range of PCcirc values (0 to 7.36) and the lack of agreed ultrastructural cutoffs for cg as a measure of CR. The testing of ultrastructural cutoffs of cg (2 or 4 affected capillaries, for instance, as opposed to the 3 used here) is beyond the scope of this article but a profitable area for future work. The 85% cumulative incidence of cpc and cg in the current study indicates that electron microscopy is of value in the diagnosis of CR, because the ultrastructural verification of cpc or cg enables the pathologist to separate CR from the broader diagnosis of chronic allograft nephropathy. Once the presence of CR has been confirmed, the causes of immune injury to the graft can be analyzed (inadequate immunosuppression, dose adjustment, patient’s compliance, a need to use better immunosuppressive drugs, etc.) Furthermore, a late graft dysfunction may be attributable to de novo or recurrent disease, or a combination of different diseases, which are difficult to recognize adequately with standard light microscopy. These reasons led us to conclude that the analysis of allograft biopsy specimens from patients with a late dysfunction by means of light microscopy, immunofluorescence, and electron microscopy provides more correct diagnoses than does light microscopic evaluation per se. There are arguments, however, that question the routine use of electron microscopy in the biopsy evaluation of a late graft dysfunction. The ultrastructural verification of CR is time-consuming, and the costs of electron microscopy are considerable. There is no specific therapy for chronic allograft nephropathy or CR. Acute rejection and cyclosporine toxicity, which require specific therapeutic intervention, can be diagnosed adequately by means of standard light microscopy. Because the therapeutic consequences remain limited in cases with diagnoses that are more correct, nephropathology units should decide individually as to whether to use light microscopy or all 3 modalities in the evaluation of graft biopsy specimens relating to a late dysfunction. In summary, the basement membrane of the PCs undergoes circumferential multilayering in several con-
ditions. We graded the change and showed that PCs with a severe lesion or 3 or more PCs with a moderate lesion were specific for CR (the cpc lesion). The cpc lesion has a characteristic appearance in semithin plastic sections. If a systematic ultrastructural search is performed for cpc and for cg, they may be identified in most of the cases involving CR. Electron microscopy is therefore a valuable tool in the morphologic diagnosis of CR. Acknowledgment. The authors thank H. E. Hansen and S. Olsen (Aarhus, Denmark) for providing the renal biopsy material on psoriatics treated with cyclosporine, and to C. Drachenberg (Baltimore, MD) for sharing the case of endstage renal disease caused by reflux nephropathy. Fruitful discussions with Kiril Trpkov (Edmonton, Canada) are appreciated. The authors appreciate the comments of the reviewers.
REFERENCES 1. Paul LC: Chronic renal transplant loss. Kidney Int 47:14911499, 1995 2. Halloran PF, Melk A, Barth C: Rethinking chronic allograft nephropathy: The concept of accelerated senescence. J Am Soc Nephrol 10:167-181, 1999 3. Paul LC: Chronic allograft nephropathy: A model of impaired repair from injury? Nephrol Dial Transplant 15:149-151, 2000 4. Nickerson P, Jeffery J, Gough J, et al: Identification of clinical and histopathologic risk factors for diminished renal function 2 years posttransplant. J Am Soc Nephrol 9:482-487, 1998 5. Legendre C, Thervet E, Skhiri H, et al: Histologic features of chronic allograft nephropathy revealed by protocol biopsies in kidney transplant recipients. Transplantation 65:1506-1509, 1998 6. Mihatsch MJ, Nickeleit V, Gudat F: Morphologic criteria of chronic renal allograft rejection. Transplant Proc 31:1295-1297, 1999 7. Yilmaz S, Taskinen E, Hayry P, et al: Protocol core biopsy as intermediate efficacy end-point in chronic kidney allograft rejection. Transplant Proc 28:491-493, 1996 8. Racusen LC, Solez K, Colvin RB, et al: The Banff 97 working classification of renal allograft pathology. Kidney Int 55:713-723, 1999 9. Monga G, Mazzucco G, Novara R, et al: Intertubular capillary changes in kidney allografts: An ultrastructural study in patients with transplant glomerulopathy. Ultrastruct Pathol 14:201-209, 1990 10. Monga G, Mazzucco G, Messina M, et al: Intertubular capillary changes in kidney allografts: A morphologic investigation on 61 renal specimens. Mod Pathol 5:125-130, 1992 11. Mazzucco G, Motta M, Segolini G, et al: Intertubular capillary changes in the cortex and medulla of transplanted kidneys and their relationship with transplant glomerulopathy: An ultrastructural study of 12 transplantectomies. Ultrastruct Pathol 18:533-537, 1994 12. Morozumi K, Oikawa T, Fukuda M, et al: Diagnosis of chronic rejection using peritubular and glomerular capillary lesions. Transplant Proc 28:508-511, 1996 13. Morozumi K, Oikawa T, Fukuda M, et al: Electron-microscopic peritubular capillary lesion is a specific and useful diagnostic indicator for chronic rejection of renal allografts showing less specific morphologic lesions in the cyclosporine era. Transplant Proc 29:8992, 1997 14. Drachenberg CB, Hoehn-Saric E, Heffes A, et al: Specificity of intertubular capillary changes: Comparative ultrastructural studies in renal allografts and native kidneys. Ultrastruct Pathol 21:227-233, 1997 15. Oikawa T, Morozumi K, Koyama K, et al: Electron microscopic peritubular capillary lesions: A new criterion for chronic rejection. Clin Transplant 13:24-32, 1999 (suppl 1) 16. Zachariae H, Kragballe K, Hansen HE, et al: Renal biopsy findings in long-term cyclosporin treatment of psoriasis. Br J Dermatol 136:531-233, 1997 17. Griffith MH, Crowe AV, Papadaki J, et al: Cyclosporin nephx-
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rotoxicity in heart and lung transplant patients. Q J Med 89:751-763, 1996 18. Iva´nyi B, Hansen HE, Olsen TS: Postcapillary venule-like transformation of peritubular capillaries in acute renal allograft rejection. Arch Pathol Lab Med 116:1062-1067, 1992 19. Rush DN, Jeffery JR, Gough J: Sequential protocol biopsies in renal transplant patients: Clinico-pathological correlations using the Banff schema. Transplantation 59:511-514, 1995 20. Bishop GA, Waugh JA, Landers DV, et al: Microvascular destruction in renal transplant rejection. Transplantation 48:408-414, 1989 21. Lajoie G: Antibody-mediated rejection of human renal allo-
grafts: An electron microscopic study of peritubular capillaries. Ultrastruct Pathol 21:235-242, 1997 22. Trpkov K, Campbell P, Pazderka F, et al: Pathologic features of acute renal allograft rejection associated with donor-specific antibody. Transplantation 61:1586-1592, 1996 23. Iva´nyi B, Thoenes W: Microvascular injury and repair in acute human bacterial pyelonephritis. Virchows Arch A 411:257-265, 1987 24. Rush DN, Karpinski ME, Nickerson P, et al: Does subclinical rejection contribute to chronic rejection in renal transplant patients? Clin Transplant 13:441-446, 1999
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the PCs in CRBx, and in 38% in CRnephr). A moderate lesion was observed in 0.6% of the PCs in NKD, 16% in CRBx, and 21% in CRnephr. Three or more PCs with a moderate lesion were encountered only in CR. A mild lesion was not suggestive of CR at all. In CRBx, 27 cases showed a severe lesion or 3 or more PCs with a moderate lesion (cpc; sensitivity: 59%). Four of the 27 cases lacked cg. The cumulative incidence of cpc and cg was 85%. In transplants with cyclosporine toxicity, the presence of cpc verified the coexistence of CR in 7 specimens. In conclusion, cpc is a specific marker of CR. The incidence of cpc increases as CR progresses. The lesion may be caused by a low-grade rejection injury to the PCs. Careful analysis of semithin sections promotes the better sampling of cpc. An ultrastructural demonstration of cpc and cg defines CR more precisely than does light microscopic evaluation per se. HUM PATHOL 31: 1129-1138. Copyright © 2000 by W.B. Saunders Company Key words: basement membrane, electron microscopy, native kidney disease, peritubular capillary, rejection, chronic allograft nephropathy. Abbreviations: CR, chronic renal allograft rejection; PC, peritubular capillary; PCcirc, mean number of circumferential basement membrane layers; cg, transplant glomerulopathy; cpc, a peritubular capillary with 7 or more circumferential basement membrane layers or at least 3 peritubular capillaries with 5 to 6 circumferential basement membrane layers.
Chronic allograft nephropathy is the major cause of renal allograft loss after the first year of transplantation. This nephropathy is a result of multiple injury/ stress factors. The immune factors involve panel-reactive antibodies, HLA match, immunosuppression, acute rejection, and drug compliance. The nonimmune factors include advanced donor age, ischemia-reperfusion injury, size mismatch, hypertension, cyclosporine trough levels, lipid disorders, recurrent disease, and so forth.1-6 The pathology of chronic allograft nephropa-
thy is nonspecific: interstitial fibrosis, tubular atrophy, unspecific arteriolopathy, unspecific glomerular changes, interstitial inflammation, lack of tubulitis, and HLA-DR–negative tubules are observed in the biopsy specimen.6 With the use of clinical and light microscopic criteria, a significant number of chronic allograft nephropathy cases can be classified into the narrower category of chronic renal allograft rejection (CR). Mihatsch et al6 recently reviewed 1,500 biopsies performed more than 6 months after transplantation. Thirty-five percent of the cases were diagnosed as chronic transplant nephropathy, 14% as CR, and 6% as acute and CR.6 The relatively low percentage of cases of CR raises the question of whether morphologic alterations caused by immunologic damage to the graft have become infrequent in the cyclosporine era, or whether standard light microscopy per se is insufficient for the diagnosis of CR in a certain number of cases involving CR. The aim of this article is to show that peritubular capillaries (PCs) undergo ultrastructural changes specific for CR (the cpc lesion), and that a search for the cpc lesion facilitates the diagnostic accuracy of CR. CR is a clinicopathologic entity6,7 characterized by a progressive decline in transplant function more than 3 months posttransplantation, with a compatible histology, and is not attributable to other known factors such
From the Department of Pathology, Albert Szent-Gyo¨rgyi Medical University, Szeged, Hungary; the Department of Laboratory Medicine and Pathology, Faculty of Medicine, University of Alberta, Edmonton, Alberta, Canada; the Department of Pathology, University of Calgary, Calgary, Alberta, Canada; the Renal Transplantation Unit, Clinics of Surgery, Albert Szent-Gyo¨rgyi Medical University, Szeged, Hungary; and the Division of Nephrology and Immunology, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada. Accepted for publication June 13, 2000. Supported in part by grants to Be´la Iva´nyi (OTKA T-025590, and ETT-588/1996-06, Budapest, Hungary). Address correspondence and reprint requests to Be´la Iva´nyi, Department of Pathology, Albert Szent-Gyo¨rgyi Medical University, Kossuth L. sgt. 40, H-6724 Szeged, Hungary. Copyright © 2000 by W.B. Saunders Company 0046-8177/00/3109-0022$10.00/0 doi: 10.1053/hupa.2000.16677
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as cyclosporine toxicity, obstructive uropathy, glomerulonephritis, hypertensive vascular disease, or diabetic nephropathy. The cardinal histopathologic features of CR are intimal fibrous thickening in arteries, and transplant glomerulopathy (cg).6,8 The diagnostic value of these features, however, is influenced by the fact that intimal fibrosis is not entirely specific (it also develops in aging, hypertension, etc.), and the incidence of cg varies in different series. Markers have therefore been sought that would enhance the accuracy of CR diagnosis. In 1990, Monga et al9 reported that in CR the basement membrane of the peritubular capillaries (PCs) undergoes circumferential multilayering and splitting, and the lesion was later proposed as an ultrastructural indicator of CR.10-15 However, unresolved questions remain as concerns the interpretation of the lesion. The criteria of focal or circumferential multiplication and the grades of circumferential multilayering were not precisely defined in the initial publications,9-13 and validation of the specificity of the lesion still requires the study of appropriate controls. Furthermore, the diagnostic sensitivity of the lesion is not known sufficiently accurately. To answer these questions, we performed an ultrastructural analysis of PCs in 169 renal biopsy specimens. Three parameters, the mean number of circumferential layers, the grade of circumferential multilayering, and the incidences of the grades, were used for a quantitative characterization of the state of the PC basement membrane in biopsy specimens involving CR and in controls. The latter group consisted of selected series of native kidney diseases, psoriatics treated long-term with cyclosporine, normal healthy individuals, acute rejection, and transplant nephrectomies caused by CR. The native kidney diseases included chronic glomerulonephritis, chronic tubulointerstitial nephritis, hypertensive kidney disease, scleroderma kidney, thrombotic microangiopathy, diabetic glomerulosclerosis, and elderly patients with renal parenchymal disease. Cases were chosen in which the clinical features or light microscopic lesions resembled the situation in CR. Because double-contoured glomerular capillary walls are a common feature of thrombotic microangiopathy and cg, we were interested in whether the basement membrane of the PCs in thrombotic microangiopathy undergoes circumferential multilayering. Aging and cyclosporine administration might influence the grade of basement membrane multilayering of the PCs. We therefore investigated the basement membrane of PCs in elderly patients and in psoriatics treated with cyclosporine. Once the diagnostic experience with the circumferential PC lesion had been acquired, PCs in transplant biopsies involving chronic cyclosporine toxicity also were studied for the possible presence of coexisting CR. MATERIALS AND METHODS Originally, the biopsy specimens were investigated by light microscopy, direct immunofluorescence (IgG, IgA, IgM, C3, C1q, and fibrinogen), and electron microscopy, accord-
ing to standard methods. The specimens from psoriatic patients were studied by light and electron microscopy. Electron microscopy was used to diagnose cg in cases with a Banff score of cg0 (double contours in ⬍10% of peripheral capillary loops in most severely affected glomerulus).8 The ultrastructural criteria of cg were at least a twofold thickening of the capillary wall as a result of the expansion of the subendothelial space by amorphous or lace-like material and the formation of new basal lamina with or without mesangial cell interposition in at least 3 capillary loops. If electron microscopy confirmed the presence of cg, the Banff score was corrected to cg0.5. At least 1 glomerulus per case was examined.
Quantitative Analysis of Circumferential Basement Membrane Multiplication The thin sections were placed on large slotted grids (2 ⫻ 1 mm), and all PC profiles around glomeruli were sampled and evaluated by one of us (B.I.) without knowledge of the Banff chronicity scores or group assignments (median, 14 profiles per case; range, 8 to 31). The capillary circumference was divided into 4 portions by the longest axis and the longest axis perpendicular to this. The multilayering of the basement membrane was regarded as circumferential if it was seen in at least 3 of the 4 portions of the capillary circumference. The site of reading of the basement membrane layers was the narrow peritubular interstitium. The change was recorded as PCs with 2 layers (Q2), with 3 or 4 layers (Q3-4), with 5 or 6 layers (Q5-6), with 7 or 8 layers (Q7-8), or with more than 8 layers (Q9). The mean number of circumferential layers (PCcirc) was calculated by dividing the total number of circumferentially layered PC profiles by the total number of PC profiles (Qtot): PC circ ⫽ 关共Q 2 ⫻ 2兲 ⫹ 共Q 3-4 ⫻ 4兲 ⫹ 共Q 5-6 ⫻ 6兲 ⫹ 共Q 7-8 ⫻ 8兲 ⫹ 共Q 9 ⫻ 9兲兴/Q tot The grade of circumferential multilayering was arbitrarily defined as mild (2 to 4 layers), moderate (5 to 6 layers), or severe (ⱖ7 layers). The focal multilayering of the PC basement membrane was regarded as abnormal if at least 3 layers were observed.
Study Groups Chronic Rejection, Biopsy Group. The biopsies were performed between 1991 and 1999 (Table 1). The biopsy specimens had to meet criteria of specimen adequacy (10 or more glomeruli with at least 2 arteries). Cases of CR were selected on the presence of fibrous intimal thickening in arteries or cg, the absence of severe hyaline arteriolopathy indicative of chronic cyclosporine nephrotoxicity, an insidious increase in the serum creatinine level, no history of infection, obstructive uropathy, recurrent glomerulonephritis, diabetes, or severe hypertension (ⱖ180/110 mm Hg). The cases met the definition of being characterized by a progressive decline in transplant function occurring more than 3 months after transplantation in the absence of any other known disease. The cases from Edmonton were chosen at random, and the cases from Szeged were consecutive. The study group comprised 46 biopsy specimens from 35 Hungarian and 11 Canadian patients. Forty-four patients received a cadaveric kidney, and 2 patients received a living related donor kidney. The patients were immunosuppressed with cyclosporine and prednisolone, or cyclosporine, prednisolone, and azathioprine. The dose of cyclosporine was 3 to 5 mg/kg/d, adjusted individually according to the serum cyclosporine level, posttransplantation period, and so forth. Before biopsy, 12 patients
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TABLE 1. Clinical Data in Chronic Rejection and Native Kidney Diseases (Median, Range)
Chronic rejection, biopsy group Chronic rejection, nephrectomy group Chronic sclerosing glomerulonephritis Chronic tubulointerstitial nephritis Hypertensive kidney disease Thrombotic microangiopathy/ scleroderma kidney Diabetic nodular glomerulosclerosis Elderly patients with renal disease
Male, Female
Age (yr)
BP, Systolic (mm Hg)
BP, Diastolic (mm Hg)
Serum Creatinine (mg/dL)
Posttransplantation Period (mo)
24, 22
43, 12-58
140, 100-180
90, 70-110
3.86, 1.79-12.5
21, 3-240
5, 6
44, 38-55
150, 85-175
95, 85-115
on dialysis
41, 9-156
6, 3
45, 35-60
170, 150-190
105, 80-140
4.07, 1.71-10.55
1, 7 5, 2
38, 18-46 52, 18-65
158, 120-205 180, 150-230
100, 80-125 105, 86-130
4.38, 1.68-11.36 4.54, 2.55-7.68
2, 6
48, 7-77
174, 155-200
100, 80-110
5.96, 0.93-13.63
4, 6
45, 24-71
170, 130-190
90, 80-110
1.68, 0.95-4.15
10, 4
72, 67-82
155, 110-210
87.5, 80-110
1.81, 0.98-5.98
Abbreviations: BP, blood pressure.
had mild to moderate hypertension. Thirty-two grafts terminated in chronic renal failure necessitating hemodialysis (median postbiopsy period: 7 months; range, 0 to 20). Twelve grafts are still functioning, with a serum creatinine level below 4 mg/dL in 6 patients. One patient died 10 months after biopsy (serum creatinine value at death: 2.72 mg/dL). Follow-up data were not available in 1 patient. Chronic Rejection, Nephrectomy Group. Intraoperative wedge biopsy specimens were studied from 11 Hungarian patients, all receiving cyclosporine and steroids, who underwent transplant nephrectomy because of CR (Table 1). The patients received transplanted cadaveric kidneys. Native Kidney Diseases. Forty-seven patients were from Hungary, and 9 patients were from Canada. In the group of chronic sclerosing glomerulonephritis, the patients showed an insidiously developed deterioration of the renal function. Four patients had marked proteinuria, with the nephrotic syndrome in 2 patients. In chronic nonbacterial tubulointerstitial nephritis, 2 cases were associated with Sjo¨gren’s syndrome, and 1 case with anterior uveitis. Hypertensive kidney disease was diagnosed by using the following criteria: a history of elevated blood pressure, normally positioned kidneys without scarring or signs of obstructive nephropathy, a negative search for a cause of secondary hypertension, normal blood glucose levels, and the morphologic demonstration of hyaline arteriolosclerosis and the absence of glomerular disease (Table 1). Morphologically, all cases with glomerulonephritis, tubulointerstitial nephritis, or hypertensive kidney disease showed marked interstitial fibrosis and tubular atrophy. Thrombotic microangiopathy occurred in 6 patients and was characterized by acute renal failure, anemia, and thrombocytopenia, with hemolysis in 4 patients, and prodromal diarrhea in 2 patients. Scleroderma kidney was observed in 2 patients; it displayed intimal thickening in arterioles and interlobular arteries, partial glomerular sclerosis, and a prominent juxtaglomerular apparatus. In diabetic nodular glomerulosclerosis, 6 patients had type 1 diabetes, and 4 patients had type 2 diabetes. The biopsy diagnoses in elderly patients were renal amyloidosis: 2 cases; primary glomerular disease: 7 cases; antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis: 3 cases; and hypertensive kidney disease: 2 cases. Normal Kidneys. The group comprised biopsy specimens from 5 Danish psoriatic patients with a normal kidney function, who underwent biopsies before long-term treatment with cyclosporine, and 15 baseline biopsy specimens from 8
living related donors and 7 cadaver donors. The baseline biopsy specimens, all from Edmonton, were obtained 1 hour after the completion of vascular anastomoses. Cyclosporine-Treated Native Kidneys. During the past decade, a considerable number of adult Danish psoriatics have been treated with long-term low-dose cyclosporine (2.5 to 6 mg/kg/d). The patients were rigorously controlled and regularly underwent biopsies to diagnose cyclosporine nephrotoxicity.16 Thirteen biopsy specimens, including 3 rebiopsies, were originally embedded for electron microscopy, and these were analyzed in the current study. The biopsy specimens were from 6 men and 4 women, treated for a median of 3 years (range, 1 to 5). At biopsy, the patients had normal blood pressure and blood sugar levels, and the median serum creatinine value was 1.06 mg/dL (range, 0.67 to 1.38). The light microscopic severity of chronic cyclosporine toxicity was assessed semiquantitatively by B.I. The degrees of arteriolopathy, interstitial fibrosis, tubular atrophy, hypertrophy of juxtaglomerular apparatuses, totally sclerosed glomeruli, and glomeruli with segmental lesions were scored. The sum of the scores expressed the severity of nephrotoxicity in the individual cases (median severity, 6; range, 2 to 9). Acute Rejection. The patients were from Canada (9 men and 2 women; median age, 39 years; range, 18 to 63) and were selected according to their Banff scores and typical clinical presentation. All were taking cyclosporine and had a good initial renal function, followed by an increase of at least 50% in the serum creatinine level versus the baseline value. After biopsy, all responded to anti-rejection therapy with a decline of at least 30% in the serum creatinine level. The median serum creatinine value at biopsy was 2.67 mg/dL (range, 1.93 to 6.48). The biopsies were performed a median of 39 days after transplantation (range, 10 to 252). The Banff acute scores8 were: v1, 9; v2, 2; g0, 10; g1, 1; i2, 7; i3, 4; t1, 2; t2, 7; and t3, 2. Chronic Cyclosporine Toxicity in Renal Transplants. PCs of 12 consecutive biopsy specimens from cadaveric transplant Hungarian patients with chronic cyclosporine toxicity (9 men and 3 women; median age, 42.5 years; range, 25 to 61) were evaluated. Cyclosporine toxicity was diagnosed on the basis of the presence of severe hyaline arteriolopathy in the biopsy specimen, an insidiously increasing serum creatinine level, and no history of diabetes, infection, recurrent glomerulonephritis, or severe hypertension. The median serum creatinine value at biopsy was 3.55 mg/dL (range, 2.5 to 5.34), and the
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median period between transplantation and biopsy was 50.5 months (range, 7 to 107). Four patients had mild hypertension. After the omission of cyclosporine, 6 patients exhibited a decrease in the serum creatinine level. The median graft survival in 5 patients was 20 months (range, 10 to 32). Six patients currently have functioning grafts with an acceptable function after a median follow-up period of 6 months (range, 4 to 10). One patient died 15 months after biopsy; his graft function was moderately impaired. The vascular and glomerular Banff chronicity scores8 were cv0, 1; cv1, 8; cv2, 3; cg0, 6; cg0.5, 3; cg2, 1; cg3, 2; ah3, 12.
Statistics Differences between the study groups were analyzed by the Wilcoxon test. Statistical significance was accepted at a probability level of 5%. The Banff chronicity scores and the PCcirc values in the biopsy group of CR were correlated by the Kendall test. The level of significance was 1%.
RESULTS Features of Biopsy Group of CR The clinical and morphologic data from the Edmonton and Szeged series are compared first. The 2 series then are combined, and the overall data are given. To facilitate reading, only relevant data are mentioned numerically. There was no significant difference between the series from Edmonton and that from Szeged in terms of posttransplantation period, or cv and cg scores. Significantly lower values were found in the Edmonton series as regards the ci and ct scores, PCcirc values, and serum creatinine levels (median value for Edmonton, 2.8 mg/dL; and for Szeged, 3.76 mg/ dL). In the combined material, the vascular and glomerular Banff chronicity scores were cv0, 5; cv1, 13; cv2, 18; cv3, 10; cg0, 11; cg0.5, 10; cg1, 15; cg2, 9; cg3, 1; ah0, 15; ah1, 18; and ah2, 13. The Banff acute scores showed superimposed acute rejection in 36 patients (acute tubulointerstitial rejection, 30; acute vascular and tubulointerstitial rejection, 6) and the suspicion of acute rejection in 8 patients. Morphology of Circumferential Basement Membrane Multilayering Normally, PCs in the cortical labyrinth consists of an extremely flat, fenestrated endothelium that rests on a thin basement membrane (Fig 1). The
severe (Figs 2 and 3) or moderate lesions (Fig 4) had tightly packed basement membrane layers, which were frequently split and usually were devoid of collagen fibers. The endothelium was nonfenestrated and had a serrated contour along the interstitial aspect of the cell body. The mild lesion contained collagen fibers within the loosely arranged basement membrane layers (Fig 5), splitting was infrequent, and the endothelium was fenestrated rather than nonfenestrated. The number of layers was usually higher along the wide interstitial aspect than along the narrow interstitial aspect of the PC. On occasion, the layers fused into a thickened basal lamina. Irrespective of the grade of the lesion, the capillary wall sometimes contained lymphocytes (observed in 16 cases). Interstitial myofibroblasts were frequently localized around the PCs. In 10 microvessel profiles from 8 specimens, an ultrastructural distinction between an efferent arteriole and a PC with a myofibroblast layer was impossible, and these profiles were excluded from further analysis. In semithin plastic sections, PCs with a moderate/severe lesion appeared as serrated profiles with a thick, ribbon-like basement membrane layer (Fig 6; compare with normal PCs in Fig 7). PCcirc Values and Incidences of Grades of PCs with Circumferentially Multilayered Basement Membrane In the biopsy group of CR, a severe lesion was recorded in 21 specimens. It was present in 3 specimens that did not display cg. PCs with a moderate lesion but without a severe lesion were noted in 16 grafts. Among these, 6 grafts had 3 or more PCs with a moderate lesion. Of the 6 grafts with 3 or more PCs with a moderate lesion, 1 graft lacked cg. A mild lesion or PCs without a circumferential multilayered basement membrane were recorded in 9 grafts. Moderate and severe multilayering occurred more frequently and extensively in the graftectomies than in the biopsy group of CR (Figs 8, 9), with correspondingly higher PCcirc values in the graftectomies (P ⬍ .01). A severe lesion and 3 or more PCs with a moderate lesion occurred exclusively in CR (the cpc lesion). Twenty-seven patients had cpc lesion (sensitivity: 59%), whereas 35 patients had cg 0.5 or greater (sensitivity, 76%). The cumulative incidence of cpc and cg was 85%. A mild lesion was not suggestive
FIGURE 1. A normal peritubular capillary from the cortical labirynth: The flat and fenestrated endothelium rests on a thin basement membrane. IS, narrow peritubular interstitium; lumen, lumen of the capillary; TBM, tubular basement membrane. (original magnification ⫻ 21,000.)
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FIGURE 2. Severe lesion: Circumferential multiplication and splitting of the basement membrane along the wide and narrow peritubular interstitium. The capillary has a serrated contour, the endothelium (END) is thickened and nonfenestrated, and the number of basement membrane layers is 7. IS, narrow peritubular interstitium; lumen, lumen of the capillary. (original magnification ⫻ 22,000.)
of CR at all. There was no significant correlation between the Banff chronicity scores and the PCcirc values (Table 2; P ⬎ .5 for all scores). In native kidney diseases, a moderate lesion was recorded in 2 PCs in 1 case of chronic tubulointerstitial nephritis (PCcirc value, 2.63). This case was not associated with the Sjo¨gren syndrome. In 2 cases of thrombotic microangiopathy (PCcirc values, 2 and 2.78), 1 and 2 PCs, respectively, displayed a moderate lesion.
Renal Transplants With Chronic Cyclosporine Toxicity Cyclosporine toxicity and CR coexisted in 7 biopsy specimens, confirmed by the presence of cpc in 7 specimens. Six of the 7 specimens also displayed cg, which was identified only ultrastructurally on 3 occasions. The vascular scores were cv0, 1; cv1, 4; and cv2, 2. In the remaining 5 cases (cv1, 4 and cv0, 1), the
FIGURE 3. Severe lesion: multiplication (at least 10 layers) and splitting of the basement membrane. Collagen fibers are absent in the area of basement membrane change. The endothelium (END) is thickened and nonfenestrated, and the lumen contains 3 lymphocytes. IS, narrow peritubular interstitium. (original magnification ⫻ 31,000.)
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FIGURE 4. Moderate lesion: Five to six circumferential layers of the basement membrane, devoid of collagen. IS, narrow peritubular interstitium; lumen, lumen of the capillary. (original magnification ⫻ 27,000.)
ultrastructural search for cpc and cg provided negative results. DISCUSSION Previous publications on circumferential multilayering of the PC basement membrane did not describe the quantitative changes adequately. This is because the PCs are understudied in renal pathology, and the methodology for assessing quantitative PC changes is undergoing development. Our study provides clues for a quantitative analysis of PCs in CR. We sampled all available PC profiles, the sampling being performed exclusively from the cortical labyrinth. It was important to exclude terminal profiles of arterioles, because the basal lamina of the arterioles in cyclosporine-treated patients shows multilayering,17 a change similar to that seen in PCs in CR. In our material, differentiation of a myofibroblast-encircled PC profile from an efferent arteriolar profile was impossible on only 10 occasions, a negligible proportion in the determination of the incidences of circumferentially multilayered PCs. The circumferential lesion was defined as seen in at least 75% of the capillary perimeter. Drachenberg et al14 defined the basement membrane multilayering as circumferential if it was seen in more than 60% of the vessel’s circumference. The stricter criterion that we used re-
duced the possibility of interpreting a focal lesion as circumferential. In a previous ultrastructural stereologic study, PCs from the cortical labyrinth were examined in renal graft biopsy specimens involving acute rejection.18 Hypertrophy of endothelial cells, a disappearance of fenestration, an increased adherence and passage of lymphocytes, and defects in the endothelial lining were demonstrated. The observations showed that during acute rejection PCs undergo a postcapillary venule-like transformation that enhances the influx of inflammatory cells into the graft. Because CR and subclinical acute rejection may coexist in biopsy specimens at 3 months or more after transplantation,19 in the current study, we decided to analyze the multiplication of the basement membrane of PCs that seemed to reflect chronic damage and was easy to read, and not to deal with endothelial cell changes that may be influenced by the acute rejection process. Reproducibility studies on the evaluation of the PC basement membrane lamination have not been performed yet. In our experience, PCs with a severe or moderate lesion cannot be misinterpreted. The serrated outline of the endothelial cells and splitting of the basement membrane, however, may be absent in PC profiles that are transitional between the mild and moderate grades. In such a situation, the evaluation must be strictly confined to the narrow peri-
FIGURE 5. Mild lesion: Collagen fibers within the 2 to 3, loosely arranged layers of the basement membrane. IS, narrow peritubular interstitium; TBM, tubular basement membrane; lumen, lumen of the capillary. (original magnification ⫻ 25,000.)
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FIGURE 6. Moderate/severe peritubular capillary lesions in semithin plastic section: The 2 capillaries (PC) have a ribbonlike basement membrane, and serrated contour. (Toluidine blue, original magnification ⫻1,000.)
tubular interstitium. If there is any difficulty in the determination of the number of layers (focal fusion of the layers, the narrow and wide interstitium cannot be recognized properly, or more layers are present on the opposite side of the narrow peritubular interstitium), the lesion should be categorized into the lower grade. If future investigations on the PCs in CR adopt this mode of evaluation, the chance of misinterpretation of the circumferential PC lesion seems low. Although fibrous intimal thickening is a strong indicator of CR, the diagnostic specificity of the lesion is reduced if the recipient has hypertension or diabetes, or the recipient received a graft from a hypertensive or aged donor. To assess whether the multilayering of the PC basement membrane is independent of the conditions listed above, for the first time in the literature we applied highly selected series of controls. In the study by Drachenberg et al,14 85 consecutive biopsy specimens with native kidney disease and 6 allografts with diseases other than cg were evaluated as controls. Their definitions of the circumferential lesion and the severe grade were similar to ours. This fortunate situation allows a combination of the results of the 2 studies in terms of the specificity of circumferential multilayering. Accordingly, the overall controls comprise 174 native kidney and 17 allograft biopsy specimens. The most important conditions include normal kidney, aging kidney, chronic cyclosporine nephrotoxicity in native kidney, hypertensive kidney disease, primary sclerosing and nonsclerosing glomerular diseases, diabetic nephropathy, amyloidosis, acute and chronic tubulointerstitial nephritis, and acute rejection. In both studies, the severe lesion was encountered only in CR, but not in the controls. A moderate PC lesion was a characteristic feature of CR in our material. However, the lesion occurred in 2 cases of thrombotic microangiopathy and 1 case of chronic tubulointerstitial nephritis in our series, and in 2 cases of thrombotic microangiopathy in the Drachenberg series. These findings indicate that thrombotic microangiopathy can be associated with a moderate degree of circumferential PC membrane multilayering. Although not documented appropriately in the literature, obstructive uropathy, analgesic nephropathy and radiation nephritis potentially might
show multilayering of the PC basement membrane in the cortical labyrinth. We recently examined the PCs in a native end-stage kidney from an adult patient with reflux nephropathy. Of the 9 periglomerular PCs studied, 1 displayed a severe lesion, 5 a moderate lesion, and 3 a mild lesion. This finding draws attention to the need to collect cases with obstructive uropathy before the end-stage phase to determine whether the condition is associated with severe circumferential multilayering of the PC basement membrane. We conclude that if obstructive uropathy, thrombotic microangiopathy, analgesic nephropathy, and radiation nephritis are excluded clinically in a graft with a late dysfunction, cpc can be used as a specific marker for CR. The sensitivity of cpc was 59%. The incidence of a severe PC lesion in the Drachenberg series was 57%.14 These figures show that PC lesions specific for CR occur in approximately 60% of the cases with CR. A 60% sensitivity may seem low as a reliable marker of CR, but it must not be forgotten that cpc can be used safely for the diagnosis of CR in those biopsy specimens in which the specificity of arterial intimal fibrosis may be questioned, or cg is lacking (we had 4 such cases). The diagnostic value of cpc was justified in our transplant patients with chronic cyclosporine toxicity, in whom the presence of cpc verified the coexistence of CR in more than half of the cases. Earlier studies did not specify how to recognize the circumferential multilayering of the PC basement membrane light microscopically. On reviewing the semithin sections, we found that the severe/moderate PC lesions had a typical appearance: a serrated outline of the endothelial cell body along the interstitial aspect of the PC, underlined by thickened, ribbon-like basement membrane material. A careful analysis of semithin sections, therefore, may promote a better sampling of cpc. The serrated contour of the PCs may be caused by the shrinkage of the microvessel. The pathogenesis of the circumferential lamination and splitting of the basement membrane of PCs is not known precisely. It may be recalled that, by express-
FIGURE 7. Normal peritubular capillaries (asterisks) in semithin plastic section from a patient with chronic rejection. (Toluidine blue, original magnification ⫻1,000.)
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FIGURE 8. Peritubular capillaries with circumferentially multilayered basement membrane occurred frequently in chronic rejection. The incidence increased, as chronic rejection progressed. Abbreviations: Normal, healthy individuals; CyA, psoriatics treated with cyclosporine; AR, acute rejection; NKD, native kidney diseases; CRBx, biopsy group of chronic rejection; Crnephr, nephrectomy group of chronic rejection.
ing HLA class I and class II molecules, PCs are the targets of rejection responses.20-22 The transcapillary passage of inflammatory cells has been shown to be associated with mild defects in the microvessel wall.23 Because circumferentially multilayered PCs were significantly characteristic of CR (P ⬍ .001 v other groups), it is likely that the development of cpc is related to rejection injury to the PCs. Our data showed that circumferentially multilayered PCs were focally distributed in the cortical labyrinth and, as CR progressed, the distribution of PCs showing a circumferential lesion broadened, and the grade of the lesion worsened. The focal nature of cpc does not favor antibody-mediated rejection mechanisms. In the current study, lymphocytes in the PC wall were noted in 16 specimens elec-
tron microscopically, and light microscopic evidence of acute tubulointerstitial rejection was recorded in 78% of the cases. Protocol biopsy specimens from patients with a clinically stable graft function showed that subclinical rejection of the graft had occurred in a significant number of cases,5,19 and untreated subclinical rejection was associated with a late graft dysfunction.24 The high frequency of concomitant acute rejection is suggestive of the idea that the cpc lesion is attributable to repeated or ongoing slowly progressive, smoldering cell-mediated rejection injury to the PCs. The target antigens may be either the classical HLA antigens or cryptic antigens exposed during previous tissue damage.3 The PCcirc values did not correlate with the grades
FIGURE 9. The severe lesion was specific, and the moderate lesion was highly suggestive for chronic rejection. The mild lesion was not indicative for chronic rejection. Abbreviations: Normal, healthy individuals; CyA, psoriatics treated with cyclosporine; AR, acute rejection; NKD, native kidney diseases; CRBx, biopsy group of chronic rejection; Crnephr, nephrectomy group of chronic rejection.
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TABLE 2.
Mean Number of Circumferential Basement Membrane Layers
Normal Cyclosporine-treated psoriatics Acute rejection Native kidney diseases Chronic rejection, biopsy Chronic rejection, nephrectomy
PCcirc ⫾ SD
Range of PCcirc
0.02 ⫾ 0.06 0.03 ⫾ 0.14 0.26 ⫾ 0.3 0.53 ⫾ 0.65 2.87 ⫾ 1.83* 5.48 ⫾ 2.02
0-0.21 0-0.5 0-0.89 0-2.78 0-7.36 2.28-8.14
Abbreviation: PCcirc, mean number of circumferential basement membrane layers. * Significantly different from nephrectomies (P ⬍ .01) and other groups (P ⬍ .001). The difference between native kidney diseases vs acute rejection was also significant (P ⬍ .02).
of cg. This finding was surprising, because cpc and cg coexisted in most of the CR cases. The lack of a mathematical correlation, however, does not automatically exclude a biologic correlation. The reasons for no correlation may include the wide range of PCcirc values (0 to 7.36) and the lack of agreed ultrastructural cutoffs for cg as a measure of CR. The testing of ultrastructural cutoffs of cg (2 or 4 affected capillaries, for instance, as opposed to the 3 used here) is beyond the scope of this article but a profitable area for future work. The 85% cumulative incidence of cpc and cg in the current study indicates that electron microscopy is of value in the diagnosis of CR, because the ultrastructural verification of cpc or cg enables the pathologist to separate CR from the broader diagnosis of chronic allograft nephropathy. Once the presence of CR has been confirmed, the causes of immune injury to the graft can be analyzed (inadequate immunosuppression, dose adjustment, patient’s compliance, a need to use better immunosuppressive drugs, etc.) Furthermore, a late graft dysfunction may be attributable to de novo or recurrent disease, or a combination of different diseases, which are difficult to recognize adequately with standard light microscopy. These reasons led us to conclude that the analysis of allograft biopsy specimens from patients with a late dysfunction by means of light microscopy, immunofluorescence, and electron microscopy provides more correct diagnoses than does light microscopic evaluation per se. There are arguments, however, that question the routine use of electron microscopy in the biopsy evaluation of a late graft dysfunction. The ultrastructural verification of CR is time-consuming, and the costs of electron microscopy are considerable. There is no specific therapy for chronic allograft nephropathy or CR. Acute rejection and cyclosporine toxicity, which require specific therapeutic intervention, can be diagnosed adequately by means of standard light microscopy. Because the therapeutic consequences remain limited in cases with diagnoses that are more correct, nephropathology units should decide individually as to whether to use light microscopy or all 3 modalities in the evaluation of graft biopsy specimens relating to a late dysfunction. In summary, the basement membrane of the PCs undergoes circumferential multilayering in several con-
ditions. We graded the change and showed that PCs with a severe lesion or 3 or more PCs with a moderate lesion were specific for CR (the cpc lesion). The cpc lesion has a characteristic appearance in semithin plastic sections. If a systematic ultrastructural search is performed for cpc and for cg, they may be identified in most of the cases involving CR. Electron microscopy is therefore a valuable tool in the morphologic diagnosis of CR. Acknowledgment. The authors thank H. E. Hansen and S. Olsen (Aarhus, Denmark) for providing the renal biopsy material on psoriatics treated with cyclosporine, and to C. Drachenberg (Baltimore, MD) for sharing the case of endstage renal disease caused by reflux nephropathy. Fruitful discussions with Kiril Trpkov (Edmonton, Canada) are appreciated. The authors appreciate the comments of the reviewers.
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