antibody - mediated rejection ( abmr ) is a recalcitrant entity with great impact on patient and graft survival [ 1 , 2 ] . in the past decade ,
improvements in hla technology along with the recognition of the role of c4d in abmr have revolutionized the understanding of this important entity , and significant advances have occurred in the treatment of abmr [ 35 ] . however , the mechanism of abmr is far from being fully elucidated , and the long - term survival of these allografts is greatly reduced when compared to that of grafts without rejection or history of t cell - mediated rejection ( tcmr ) [ 2 , 6 ] .
microcirculation inflammation , including glomerulitis and peritubular capillaritis ( ptcitis ) , has been recognized as a cardinal feature in the diagnosis of abmr [ 9 , 10 ] .
peritubular capillary dilation is another important side of microcirculation changes , and although it has been noticed in the abmr for years , it is far from being clearly demonstrated and its pathogenesis remains unclear .
t - bet is a member of the t - box family of transcription factors regulating th1 lineage commitment . in a recent study
, we found that transplant glomerulopathy , a principal form of late abmr , had a significant increase in t - bet expression in peritubular capillaries ( ptc ) , and this expression was strongly correlated with the count of intra - ptc inflammation cells .
furthermore , ptc dilation was also strongly correlated with the intra - ptc inflammation . in a previous study
, we found intraglomerular inflammation correlated with in situ expression of t - bet in patients with abmr .
we hypothesize that t - bet expression might be also correlated with ptc injury in early abmr .
hif-1 is a transcription factor which acts as a master regulator coordinating oxygen homeostasis , and the hif system is ubiquitous which is instantaneously up - regulated upon hypoxia . in abmr , whether ptc injury can cause tissue damage via hypoxia is unknown . in this study
, we explored the dilation of ptc in relation to inflammation , t - bet expression , and hypoxia .
the patients were retrospectively selected from among 226 renal allograft recipients who had performed renal biopsy between june 2008 and may 2012 at jinling hospital , nanjing university school of medicine , nanjing , china . among them , 18 recipients were diagnosed as having c4d - positive acute rejection episodes according to clinical manifestations and histological features .
the diagnosis was based on the following : ( 1 ) clinical evidence of acute rejection , manifested as rapid renal dysfunction and/or decrease of urine volume ; ( 2 ) c4d deposition in the ptc area ; and ( 3 ) pathologic features that met banff 's 1997 criteria for acute rejection grade i , ii , or iii .
additionally , rejection episodes occurring beyond the first month after transplantation are more likely to offer a mixed histologic picture , often demonstrating acute and chronic , vascular and tubulointerstitial , pathology .
this group was compared with a group of tcmr patients who were diagnosed within the same time period . since ptcitis ( ptc ) and glomerulitis ( g )
are often associated with abmr and g + ptc = 0 was confirmed to be a useful diagnostic algorithm for tcmr exclusion , we excluded all the recipients with ptcitis and glomerulitis in tcmr group .
these patients were randomly matched with a group of recipients with stable graft function who received protocol biopsies as controls .
in addition , we also included a tg group to compare ptc variation with abmr .
we applied the following inclusion criteria : ( 1 ) biopsy confirmation of the presence of a duplication of the glomerular basement membrane on periodic acid - schiff or silver stain , and ( 2 ) 1 year of follow - up after the diagnosis .
an electron microscopic evaluation was performed to exclude membrane duplication that was caused by recurrent or de novo glomerular disease .
informed consent was obtained from all patients , and the human subjects committee of jinling hospital , nanjing university school of medicine approved all of the study protocols .
protocol biopsies were performed between days 12 and 17 posttransplantation ( so - called 2-week protocol biopsy ) , and diagnostic biopsies were performed upon clinical indication and according to local standard of practice .
two needle biopsy cores were obtained from each renal allograft for morphologic study : one for formalin fixation and the other for quick - freezing .
hematoxylin and eosin , periodic acid schiff , methenamine - silver , and masson stains were routinely used on the formalin - fixed tissue .
fresh - frozen tissues were analyzed by immunofluorescence microscopy using a conventional panel of antibodies against igg , igm , iga , c3 , c4 , c1q , and c4d .
c4d staining was routinely performed on frozen slides using an indirect immunofluorescence technique with a primary affinity - purified monoclonal antibody ( mouse anti - human ; quidel , san diego , ca , usa ) and an fitc - labeled affinity - purified secondary rabbit anti - mouse igg antibody ( dako , denmark ) .
positive c4d staining was defined as a bright linear stain along the capillary basement membranes that involved over half of the sampled capillaries in accordance with the 2001 banff meeting .
cd4 , cd8 , cd68 , and cd20 were regularly detected when a biopsy was performed .
the intragraft expression of hif-1 and cd31 was retrospectively studied via immunohistochemistry using stored residual biopsy tissues .
the antibody regimens were conducted as follows : mouse monoclonal antibody hif-1 ( novus biologicals , littleton , co , usa ) ; mouse monoclonal antibody against t - bet ( santa cruz biotechnology , santa cruz , ca ) ; and mouse monoclonal antibodies against cd4 ( novocastra , newcastle upon tyne , uk ) , cd8 ( novocastra ) , cd68 ( dako , carpinteria , ca , usa ) , cd20 ( dako ) , and cd31 ( dako ) .
the sections were reviewed by two pathologists , and the results are expressed as the total number of positive cells per glomerulus or per square millimeter in the cortex . to assess the number and size of ptcs , a cd31 costaining was used as previously described [ 18 , 19 ] .
the ptc density was calculated by counting the total number of ptcs within the confines of each of 10 random 0.25  mm fields ( each of these fields was delineated by a 1  cm ocular grid that was viewed at 400 magnification ) , and the result is expressed as the mean per field .
the diameter of these ptcs was also measured , and their mean was calculated in micrometers . to measure the ptc spaces , slides from each case
were examined using an olympus ix70 inverted system microscope ( olympus america , melville , ny , usa ) connected to a hewlett packard computer with image - pro plus software ( media cybernetics , silver springs , md , usa ) .
the intra - ptc area is expressed as the proportion of total ptc spaces over the entire cortex field .
intraglomerular capillaries were measured for their quantity ( capillary counts per glomerulus ) and diameter on cross - section of the glomeruli . to calculate the intracapillary inflammation cells , costaining of cd31 and inflammation markers ( e.g. , cd4 , cd8 , and cd68 ) were performed , and the results are expressed as the mean per ptc or glomerulus .
glomerular cross - sectional area was also determined using the image - pro plus software .
statistical analyses were conducted using spss ( v16.0 ) and graphpad prism ( v5 ) software .
pairwise comparisons of variables based on proportions were done by fisher 's exact test with bonferroni correction for p value .
continuous variables were presented as mean  s.d . and compared using one - way analysis of variance ( anova ) followed by post hoc pairwise comparisons using lsd tests , or analyzed using nonparametric method if the data were not normally distributed . ordered categorical data
were presented as median ( 25th75th percentiles ) and compared using the nonparametric kruskal - wallis anova on ranks for global comparison , followed by duncan 's analysis for multiple comparisons .
the level of statistical significance was set at p  0.05 ( two - sided ) .
forty - five renal allograft recipients were included in this study , including 18 cases of abmr , 13 cases of tcmr , and 14 cases of stable grafts as controls .
there were no significant differences among the three groups with respect to patient age , gender , time of prior transplantation , time of biopsy , or incidence of positive panel - reactive antibody .
each patient received anti - il-2 receptor monoclonal antibody for the induction of immunosuppressive therapy and was subsequently maintained on a similar immunosuppressive protocol after transplantation ( table 1 ) .
a comparison of histological lesions in the three groups is given in tables 2 and 3 .
recipients with lymphocytes infiltration in ptcs or glomeruli were excluded from tcmr group while ptcitis was observed almost in all patients in abmr group .
tubulitis was seen in all patients of acute rejection enrolled in the study , compared with 42.9% of stable graft .
significantly more patients ( 88.9% ) have intimal arteritis in the abmr group compared with either of the other two groups .
we used immunohistochemistry to detect cd4 , cd8 , cd68 , and hla - dr expression . in the abmr group , the average values for cd4 , cd8 , cd4/cd8 and cd68 , were all similar to the tcmr group .
however , when compared with stable grafts group , every kind of lymphocyte is significantly higher in both abmr and tcmr groups . labeling the endothelial cells for cd31 made it possible to calculate the number and diameters of the capillaries .
figures 1(a ) and 1(b ) show that cd31 staining labeled the capillaries notably well .
overall , abmr was correlated with an increased ptc diameter and a decreased ptc density .
we compared the ptc density and capillary diameter among the three groups ( figures 1(c ) and 1(d ) , table 4 ) . in the abmr group ,
the density of ptcs was significantly lower compared with the stable graft group ( 22.22  2.51 versus 25.64  1.82/field , resp .
, p < 0.001 ) , whereas the diameter of the existing ptcs was significantly larger ( 2.34  0.81 versus 1.09  0.29  m , resp .
, p < 0.001 ) , suggesting a rarefaction and dilation of the capillaries ( figure 1(a ) ) . however , in the tcmr group , we found no such rarefaction and dilation of ptcs as observed in the abmr group ( figure 1(b ) ) .
the capillary density was similar between the tcmr group and protocol biopsies ( 27.23  2.49 versus 25.64  1.82/field , resp .
in addition , image - pro plus revealed a significantly larger intra - ptc area in the abmr group ( 0.035  0.018 ) compared with the tcmr ( 0.023    0.012 , p = 0.026 ) and stable graft ( 0.020  0.006 , p = 0.006 ) groups ( figure 1(e ) ) .
in addition to ptcs , capillary dilation was also observed within the glomeruli . however , there were no significant differences between the three groups in the diameter of the intraglomerular capillaries and intraglomerular capillaries ' area .
in the abmr group , we detected a significant correlation between ptc dilation and microcirculation inflammation .
the dilation of ptcs was strongly correlated with the quantity of intra - ptc infiltrating cells ( r = 0.664 , p = 0.004 ) , as shown in figure 2(a ) .
in contrast , as shown in tcmr group ( figure 1(b ) ) and sg group ( not shown ) , capillary dilation was rare , and it was also found to mostly coexist with luminal inflammatory cells .
we also calculated the ratio of the total intra - ptc area within the cortex to the area of the entire cortex in abmr group , and found that this ratio was also strongly correlated with intra - ptc cell counting ( r = 0.578 , p = 0.006 ) ( figure 2(b ) ) .
a significant increase in t - bet expression was detected in the ptcs , and the majority of t - bet+ cells were typically located within the capillary lumen .
intra - ptc t - bet expression was also detected in the tcmr group ; however , its expression was much lower than that in the abmr group ( 1.34  0.56 versus 0.15  0.22/capillary , p < 0.001 ) ( figure 3(a ) ) . in the abmr group , the t - bet expression correlated well with the quantity of infiltrating cells ( r = 0.768 , p < 0.001 ) ( figure 3(b ) )
moreover , since ptc dilation is correlated with microcirculation inflammation as above mentioned , we also find a good correlation between the intra - ptc expression of t - bet and the ptc diameter ( r = 0.491 , p = 0.038 ) ( figure 3(c ) ) .
similar to the findings in experimental acute renal failure , the strongest and most abundant signals were detected in the medulla , and signal intensity and abundance increased from the outer medulla to the deep papilla . moreover ,
cortical tubular immunostaining for hif-1 was positive and even signals can be detected in some glomeruli . according to the results of double staining of cd31 and hif-1 , capillary dilation and hif-1 expression occurred somewhat in parallel both in abmr and tcmr .
it is noteworthy that , as shown in figure 4 , ptc dilation could be observed where hif-1 was up - regulated while hif-1 expression was not necessary for capillary dilation . in order to compare the capillary variation between abmr and tg , 32 cases of tg were selected as control group ( the baseline characteristics previously described in ) .
we found that the ptc loss is similar in two groups , while the ptc diameters were significantly larger in tg group ( 2.34  0.81 versus 6.35  2.16 , p < 0.001 ) , leading to much more severe ptc dilation .
moreover , the diameters of capillary loops within glomerulus were also increased , and the intraglomerulus area was also significantly increased and showed an enlarged glomerulus .
anyway , the sizes of glomerulus as well as capillary loops within glomerulus were similar between abmr and tcmr cases .
we confirmed that ptc dilation is correlated with microcirculation inflammation , and ptc inflammation is in turn strongly correlated with in situ t - bet expression .
these data suggest that t - bet plays an important role in the pathogenesis of microcirculation injury in abmr .
these findings shed a new light on the pathogenesis of abmr . though it is the general belief that abmr is associated with peritubular capillary dilation [ 2325 ] , it is not until this study that dilation has been demonstrated clearly in this setting .
our study clearly demonstrates that the ptc diameter and intra - ptc area were significantly increased in the abmr . shortly after the development of abmr ,
the diameters of ptcs are increased to twice of the control group , which leads to significantly increased intra - ptc area .
nevertheless , the dilation is diffused and can be seen in most of the ptcs , no matter in cortex or medulla .
this variation can be regarded as a feature of abmr as it can not be observed in tcmr . as the diameter of ptc is easy to measure and evaluate throughout the whole specimen , it may be applicable to biopsy interpretation and may be of good diagnostic utility for abmr .
moreover , our data also revealed a rarefaction of ptc during abmr . to our knowledge , this is the first report on ptc loss when abmr occurs .
although the diameter of each ptc is increased , the count of ptc is significantly decreased ( figure 1 , table 4 ) which can be observed in early biopsies immediately after the abmr occurs .
the quick rarefaction of ptc seems to be unique in early abmr as it is not observed in tcmr or tg .
ptc loss has been demonstrated in models involving ageing , cyclosporine , angiotensin ii infusion , chronic catecholamine infusion , glomerulonephritis , radiation - induced injury , and potassium depletion , which mostly are involved in a local alteration in the balance between angiogenic and angiostatic factors [ 27 , 28 ] .
however , the quick ptc rarefaction during the rejection suggests a dramatic process , which is most probably caused by direct damage to the capillary wall .
ptc loss after renal transplantation usually associates with increased interstitial fibrosis / tubular atrophy , and predicts reduced renal function ; thus the dramatic ptc loss during abmr is an important process to graft dysfunction .
the pathogenesis of ptc variation during abmr is not clear ; however , as the dilation can be observed quickly when abmr occurs , it should be a response to the antibody activity .
our data revealed that the ptc dilation is strongly correlated with intra - ptc inflammation .
although interstitial inflammation might cause quick vessel dilations in the early phase , the ptc dilation is more likely to be related to intra - ptc inflammation , as the dilation can not be observed in tcmr cases , which have more severe interstitial inflammation .
moreover , the dilation of the ptc lumen was strongly correlated with the degree of microcirculation inflammation , whereas ptc dilation was mostly observed in areas with ptc inflammation .
this phenomenon can be seen in tg as well and it is quite likely that the capillary dilation is caused by in situ inflammation .
those data suggest a central role of inflammation in the development of microcirculating variation during abmr .
furthermore , we find that intra - ptc inflammation is strongly correlated with in situ expression of t - bet .
this correlation is unique to the abmr group , as the tcmr and stable graft groups , which rarely have microcirculation inflammation , only rarely exhibited t - bet expression in the ptc areas .
t - bet is a member of the t - box family of transcription factors and is a key determinant of t - helper cell differentiation into th1 .
our previous study showed that t - bet expression is correlated with glomerulitis in abmr , further study revealed that both glomerulitis and ptcitis are correlated with t - bet expression in tg , which is a chronic form of abmr .
this study proved that both kinds of microcirculating inflammation , ptcitis and glomerulitis , are correlated with t - bet expression , no matter in acute abmr or chronic type .
higher t - bet expression is correlated with more ptc inflammation , and in turn is correlated with larger ptc diameters .
this study suggests that t - bet pathway might be a potential target in the management of abmr . as capillary damage
may lead to local hypoxia , it is possible that hypoxia is involved in the pathogenesis of abmr . at experimental level
, there has also been described an intense dilation of the peritubular capillaries in chronic allograft dysfunction [ 26 , 30 ] , and the morphological changes were thought to be related to chronic ischemia .
we hypothesized that hypoxia takes part in the development of ptc dilation . since hif allows for hypoxia detection at a single cell resolution [ 3133 ] , and
as previously described in vivo , renal hif-1 immunostaining was almost exclusively found in tubular segments , hif-1 immunostaining had been performed in this cohort .
somewhat surprising , ptc dilation could be observed where hif-1 was up - regulated while some dilation had nothing to do with hif-1 expression , which suggests that hypoxia is not necessary for ptc dilation in abmr .
obviously the ptc dilation in abmr has a pathway that is different to chronic allograft dysfunction .
current data also showed that the microcirculating variation in abmr is similar to its chronic pattern , tg .
both abmr and tg have significant ptc dilation , which is correlated with ptc inflammation , and in turn with in situ t - bet expression .
it suggests that both acute and chronic patterns of humeral rejection share a common mechanism of ptc variation ; t - bet expression might account for the development of microcirculation inflammation .
however , in tg , the ptc dilation is much more severe than in abmr , and there are enlarged glomerulus loops and glomerulus size , which are not shown in the abmr group .
the diagnosis of abmr has largely depended on the c4d staining in the past decade . however , in spite of high specificity , c4d is lacking sensitivity , and many cases of rejection with anti - hla are c4d negative [ 7 , 3537 ] .
the variation of ptc during abmr , including ptc dilation and rarefaction , differs to tcmr and may be used in the diagnosis of abmr .
moreover , our previous study had reported that the predominance of t - bet over gata-3 may distinct abmr from tcmr ; the current data even showed that the predominance of t - bet expression in ptc is a feature of abmr and can also be regarded as a diagnosis marker . in summary
, this study shows that abmr is associated with ptc dilation and the latter is correlated with microcirculation inflammation , and microcirculation inflammation is strongly correlated with in situ t - bet expression .
these results suggest that inflammation may take part in the pathogenesis of ptc dilation , and they warrant further investigation .
 antibody - mediated rejection ( abmr ) remains one of the major causes of graft loss after renal transplantation . 
 it is dominated by endothelial damage in microcirculation . 
 clarifying the mechanism of microcirculating damage is obviously a key step to understand the pathogenesis of abmr . 
 here we characterized capillary variation in abmr and its possible mechanisms . compared with t cell - mediated rejection and stable grafts 
 , there was a significant dilation and rarefaction in peritubular capillaries ( ptcs ) of the abmr group ; image - pro plus revealed a significantly larger intra - ptc area . 
 interestingly , the dilation of ptcs was strongly correlated with the intra - ptc cell counting . moreover , 
 peritubular capillary inflammation is correlated with in situ t - bet expression , and there was a good correlation between the intra - ptc expression of t - bet and the ptc diameter . 
 hif-1 
 up - regulation could be observed in abmr but it was not necessary for capillary dilation . in general , abmr is characterized with early capillary dilation and rarefaction ; our data confirmed that the dilation is strongly correlated with intracapillary inflammation , which in turn is correlated with in situ t - bet expression . 
 t - bet plays an important role in the development of microcirculating injury , and thus it is a potential target for the treatment of abmr . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion