Key Words: Acute rejection, ATN, Immunohistology, Morphometry, Renal allograft
Correspondence and Galley Proofs to: Niels Marcussen, M.D. University Institute of Pathology Ćarhus Kommunehospital DK-8000 Ćarhus C, Denmark Reprint requests to: Kim Solez, M.D. Department of Pathology 5B4.02 W.C. Mackenzie Health Sciences Centre University of Alberta Edmonton, Canada T6G 2R7
In acute interstitial nephritis in the native kidney, we have previously shown that tubulitis is most commonly seen in the distal part of the nephron involving the pars recta of the proximal tubules and segments distal to this . In acute rejection the distal convoluted tubules has been shown to be most severely infiltrated with lymphocytes . Leu-7 positive cells in tubules have been found to indicate rejection in the renal graft [5, 6].
The aim of the present study was to investigate and quantitate the inflammatory infiltration and the tubular changes in ATN in the native kidney and the graft and in acute rejection in order to see whether marked light microscopical differences are present. Moreover, we estimated the amount of tubulitis and determined what tubular segments are involved to see whether these features correlated with the extent of interstitial inflammatory infiltrates.
One slide from each biopsy was incubated with monoclonal antibody against MB1 , washed, rabbit anti-mouse immunoglobulins were applied for half an hour followed by mouse APAAP complex (Dakopatts 1:25). For visualization Fast Blue BB and naphtol AS-MX-phosphate substrate were used. Mouse antibodies against UCHL1  were applied for one hour on the MB1 stained slides, and then incubated with PO-labeled swine anti-mouse immunoglobulins for half an hour and stained with diaminobenzidine. The slides were counterstained with PAS. MB1 is found on all B cells excluding mature plasma cells and on some mature T cells (suppressor inducer CD4+ cells) [12, 14]. UCHL1 is a marker of T lymphocytes [15, 16].
Seven biopsies from patients with graft ATN and 10 from acute rejection were stained for Leu-7 positive lymphocytes and counterstained with Mayer's hematoxylin.
The EMA and LCA and the THP and LCA stained biopsies were semiquantitatively scored from 0 to +3 for a number of changes. The overall interstitial infiltration by LCA positive cells was scored. The localization of these cells around glomeruli, larger vessels, EMA positive tubules, EMA negative tubules and THP positive tubules were semiquantitatively estimated. The infiltration by LCA positive cells of these structures was scored as well. In those biopsies where both medullary tissue and cortex were present separate scores for changes in the cortex and in the medulla were performed. A score of 0 was given when no cellular infiltration was found, and +3 when severe infiltration of the interstitium, tubules, vessels or glomeruli were noted.
Since the maximal number of inflammatory cells per tubular cross section is an important component for the criteria of tubulitis in the Banff classification of kidney transplant pathology , the maximal number of LCA positive cells in EMA positive and EMA negative tubular cross sections was calculated in the allograft biopsies.
The intensity of MB1 and UCHL1 in the interstitium was semiquantitatively scored from 0 to +3. In the biopsies from patients with acute rejection where arteritis was found in the MB1 and UCHL1 stained sections, it was qualitatively assessed which type of lymphocytes (MB1 or UCHL1) was present in the intimal lesion.
The 17 slides stained for Leu-7 were investigated for the presence of Leu-7 positive intratubular lymphocytes.
Stereological investigation. Cortex was defined as part of the kidney where glomeruli are present. Medulla was, if present, investigated separately. Interstitial capillaries were included in the interstitium as were tubular basement membranes and Bowman's capsules. Glomeruli were defined as the glomerular tuft. In the estimation of the volume fractions of LCA positive cells both the cell nucleus and cytoplasm were included.
At a magnification of 900X point counting was used to estimate the volume fractions of EMA negative cells (proximal cells), EMA negative tubular lumens, EMA positive cells (distal nephron), EMA positive tubular lumens, interstitium, LCA positive cells in the interstitium, glomerular tuft and vessels in the EMA and LCA stained sections. The THP and LCA stained sections were used to estimate the volume fraction of THP positive cells (distal straight tubules) and THP positive tubular lumens at the same magnification.
At a higher magnification (2300X) the volume fractions of LCA positive cells in EMA positive and EMA negative tubules were estimated in five 1-hour biopsies and in all the biopsies from transplant patients with ATN or acute rejection. Point counting and a grid with two set of points were used. The ratio between the two set of points was 1/9. The number of points from the point set with the largest distance between individual points that hits the reference volume, which was EMA positive or EMA negative tubules was counted. The finer point set was used to count the number of LCA positive cells that had invaded the tubular basement membrane. From these two counts the volume fraction of inflammatory cells infiltrating either EMA positive or EMA negative tubules could be calculated. Lymphocytes in tubular lumens were not counted. The cortical labyrinth and the outer stripe of the outer medullary zone including medullary rays were investigated separately.
Statistics: Comparison between the groups was first done using the ANOVA analysis followed by unpaired t-tests. Linear regression analysis was used for correlative analysis of data. Statistical significance was accepted at the 0.05 level.
Lymphocyte markers: The immunostaining for MB1 and UCHL1 was of nearly equal intensity (Table 2). More immunostained cells were as expected found in the acute rejection group. In that group, seven cases had arteritis in the MB1 and UCHL1 stained sections and the investigation showed that in one case UCHL1 positive cells only were found in the intimal lesion and in the remaining six cases both UCHL1 and MB1 positive cells were found with a heavy predominance of UCHL1 positive cells.
Tubulitis: As expected, tubulitis was more prominent in acute rejection than in the other groups (Table 3) [Fig. 1 and 2]. The greatest difference between acute rejection and the other groups was found in the proximal tubules where the score for tubulitis was nearly as high as it was in the EMA positive tubules. In all groups, infiltration of tubules was strongest in EMA positive tubules, followed by EMA negative (proximal) tubules and less in THP positive tubules (Table 3). The average maximal number of LCA positive cells in both EMA negative and positive tubular cross sections in the 1-hour biopsies was about 1. In the graft ATN group the maximal number of LCA positive cells were 3.4 ± 1.5 and 1.8 ± 1.1 in the EMA positive and EMA negative tubules, respectively, a highly significant difference (p=0.0002). The maximal number of cells in the acute rejection group was 7.1 ± 3.6 and 4.5 ± 3.5 for EMA positive and EMA negative tubules, respectively (p=0.02). A significant correlation was found between the semiquantitative score of tubulitis in general and the semiquantitative score of LCA positive cell infiltration [Fig. 3].
There was a tendency toward a more severe tubulitis in the medullary rays than in the cortical labyrinth but no significant difference was found (Table 4).
Leu-7 positive intratubular cells in the graft: Of the seven cases with ATN none had intratubular Leu-7 positive cells and of the 10 cases with acute rejection 9 had Leu-7 positive cells in the tubules, a statistically significant difference with Fishers exact test.
Morphometry of renal structures: No difference was found between the groups regarding volume fraction of the glomerular tuft (Table 5). Both proximal tubules and EMA positive tubules had a significant decrease in volume fraction in both transplant groups compared with the 1-hour biopsies. In the transplants, the most marked decrease was found in the acute rejection group. The total interstitium was increased in all three groups compared with the controls, but most in the group from patients with acute rejection, where also the largest infiltrations with LCA positive cells were seen. A significant correlation was found between the semiquantitation of LCA positive cell infiltrates and the volume fraction of these infiltrates (r=0.85, p<0.0001 [Fig. 4]).
Correlation between tubulitis and interstitial infiltration in biopsies with acute rejection: A significant correlation was found between the semiquantitative estimates of tubulitis in the proximal tubules and both the semiquantitative and morphometric estimates of LCA infiltration [Fig. 5]. No such correlation was found regarding the EMA and THP positive tubules (data not shown).
No significant correlation was found between the semiquantitative score of tubulitis in the different tubular segments and the score of UCHL1 cells. For MB1 cells a positive correlation was found between the interstitial infiltration of MB1 cells and the score of tubulitis in THP positive tubules (r=0.64, p<0.05).
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