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- 2011 USCAP Meeting Abstracts (1)
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Early Ultrastructural Abnormalities of Transplant Glomerulopathy
(TG): Correlation with C4d-Positive and C4d-Negative Antibody-Mediated
Rejection (AMR) and Subsequent Development of Overt TG.
Mark Haas. Cedars-Sinai Medical Center, Los Angeles, CA
Background: TG is correlated with reduced renal allograft survival and with donor-specific antibodies (DSA). Overt TG, with double contours of the glomerular basement membrane (GBM) on PAS and silver stains, is typically seen >1 year post-transplantation (PT). However, electron microscopic (EM) changes correlated with development of overt TG may be seen much earlier; these include glomerular endothelial swelling, subendothelial electron-lucent widening, and early GBM duplication [Wavamunno et al, AJT 7: 1-12, 2007]. This study aims to examine the specificity of these early EM changes for AMR, both C4d-positive and C4d-negative, and determine if these are inevitably associated with later development of overt TG.
Design: From 1/07 – 12/09, 119 renal allograft biopsies were done within the first 3 months PT on patients followed at our center; of these 95 (from 91 patients) were examined by EM. The remaining 24 had inadequate tissue for EM or were repeat biopsies. The 95 biopsies form the study group; indirect immunofluorescence for C4d was done on all 95, and DSA data at the time of biopsy was available for 69.
Results: Of the 95 biopsies, 12 showed C4d+ AMR with glomerulitis and/or peritubular capillaritis (Banff g + ptc ≥2), peritubular capillary C4d (diffuse in 11), and DSA; 2 also had Banff type 1 cellular rejection (ACR). 7 biopsies showed histologic changes of AMR with DSA, but no C4d (C4d- AMR); 4 had type 1 ACR. 21 additional biopsies had type 1 (16) or type 2 (5) ACR; 55 had no diagnostic rejection. One or more early EM changes of TG were seen in 12/12 biopsies with C4d+ AMR, 6/7 with C4d- AMR, 8/21 with ACR (2 with histologic changes of AMR, but no DSA data), and 6/55 with no rejection. All 3 early EM changes of TG were seen in 7/12, 4/7, 3/21, and 0/55 biopsies, respectively. 14 patients (including 5 with C4d+ AMR and 4 with C4d- AMR) with ≥1 early EM changes had ≥1 follow-up biopsy 3.5 – 19 months PT. Of these 5/8 patients with persistent histologic changes of AMR and 0/6 without developed overt TG. All 5 patients with C4d+ AMR were treated for AMR and 1 developed overt TG; by contrast 0/4 patients with C4d- AMR were initially treated for AMR and 3/4 developed overt TG.
Conclusions: Early EM changes of TG are seen in most cases of C4d+ and C4d- AMR and are often associated with development of overt TG, especially if there are presistent histologic changes of AMR. However, this progression does not appear to be inevitable, at least during the first 2 years PT.
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Developing a Molecular Diagnosis of Antibody Mediated Rejection: Can Molecular Assessments Replace C4d?
Banu Sis, Luis Hidalgo, Gunilla Einecke, Michael Mengel, Jeff Reeve, Konrad Famulski, Arthur Matas, Bert Kasiske, Bruce Kaplan, Philip Halloran. University of Alberta, Edmonton, Canada; University of Minnesota, Minneapolis; University of Arizona, Tucson
Background: When microcirculation lesions and HLA-antibody were used to define antibody-mediated rejection (ABMR), 63% of late kidney failures were attributable to ABMR, but many were C4d negative, suggesting that detection of this phenotype requires new diagnostic criteria (AJT 2009;9:2520-31). We previously showed that high endothelial gene set expression in kidney transplant biopsies with donor specific antibody (DSA) indicates active ABMR and predicts poor graft survival (AJT 2009;9:2312-23).
We hypothesized that measurement of individual endothelial transcripts in biopsies is a sensitive and specific method to diagnose ABMR.
Design: We studied 403 kidney transplant biopsies for cause from 315 patients by histopathology, C4d staining (368/403), antibody testing (355/403), and microarrays. ABMR was defined by histology/serology: microcirculation lesions (g>0 and/or ptc>0 and/or cg>0) plus DSA: Of 403 biopsies, 24 had C4d+ ABMR, 45 C4d negative ABMR, and 14 mixed rejection. The new molecular/serology definition of ABMR included high endothelial gene expression in the presence of DSA.
Results: We evaluated expression of three endothelial transcripts: SOX7 (SRY-box 7), CDH5 (cadherin 5), and VWF (von Willebrand factor). In a training set (n=201), we selected a cut-off signal for each gene for detecting ABMR (histology/serology) with at least 80% sensitivity by ROC curve analysis.
Applying the same cut-offs in an independent set (n=202), SOX7 plus DSA showed 88% sensitivity and 90% specificity, and combined SOX2/CDH5/VWF plus DSA showed 90% sensitivity and 86% specificity for ABMR, whereas, C4d showed 49% sensitivity and 96% specificity.
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Dystroglycan Patterns in FSGS Variants.
Giovanna Giannico, Sharon Phillips, Yu Shyr, Charles E Alpers, Vivette D D'Agati, Agnes B Fogo. Vanderbilt University, Nashville, TN; University of Washington, Seattle; Columbia University, New York, NY
Background: Background: The dystroglycan (DG) glycoprotein complex links podocytes to the glomerular basement membrane, and through interaction with utrophin interacts with actin in the cytoskeleton. Decreases in αDG have been variably reported in minimal change disease (MCD). αDG contains sialic acid, a necessary component for maintenance of foot processes and the filtration barrier. We investigated whether αDG was altered in variants of FSGS, classified according to the Columbia Classification.
Design: 69 biopsies (5 cellular, 17 collapsing, 3 perihilar, 18 tip, 19 NOS, 7 MCD) with a total of 1289 glomeruli were investigated. Each glomerulus was scored for αDG (0-3 intensity) in the lesional and non-lesional segments in involved glomeruli and in uninvolved glomeruli without segmental lesions.
Results: A mix of lesions were present in biopsies, with 39 glomeruli with cellular lesions, 401 with collapsing lesions, 296 with NOS lesions, 45 with perihilar lesions and 329 with tip lesions. αDG staining was decreased in uninvolved glomeruli in NOS (intensity 2.75), tip (2.79) and minimally decreased in cellular variant cases (2.86), with least staining in uninvolved glomeruli in collapsing glomerulopathy (intensity 2.60). Uninvolved glomeruli showed unaltered αDG in biopsies with perihilar FSGS and in MCD (3.00). Segmental lesions showed nearly absent staining in collapsing glomerulopathy (0.12), with similar low level staining in NOS lesions, and slightly more staining in tip lesional areas (1.00).
Conclusions: αDG staining is decreased in lesional areas of segmental sclerosis, and particularly decreased even in uninvolved segments of glomeruli in collapsing glomerulopathy. We speculate that this may reflect dedifferentiation of podocytes. Furthermore, these findings indicate that decreased dystroglycan may not be specifically or uniquely related to the podocyte perturbations underlying minimal change disease.
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Identification of a Potential Marker of Glomerular Fibrogenesis in Childhood Nephrotic Syndrome.
Mariana M Cajaiba, Rose Ayoob, Ronald Houston, Sheldon I Bastacky, Peter Baker. Nationwide Children's Hospital, Columbus, OH; University of Pittsburgh, PA
Background: Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are the major causes of pediatric nephrotic syndrome (NS). Most MCD cases respond to steroids, whereas FSGS tends to be resistant and progress to global sclerosis leading to chronic renal disease. The reversible nature of MCD is thought to reflect transient podocyte damage due to increased circulating cytokines, whereas FSGS would correspond to irreversible podocyte damage with activation of TGF-β-mediated apoptosis and fibrogenesis. Markers of fibrogenesis that could help to define the pathogenesis and prognosis in these cases have not been clearly identified. One of the downstream targets of TGF-β signalling is Sox9, a transcription factor involved in normal skeletal development and pathological fibrogenesis. Recent experimental studies showed increased levels of both Sox9 and TGF-β mRNA in FSGS, and increased glomerular collagen IV accumulation triggered by TGF-β-induced Sox9 expression. The aim of this study is to investigate Sox9 as a potential indicator of glomerular fibrogenesis in NS.
Design: 29 renal needle biopsies performed in children with NS were selected; 15 were diagnosed as FSGS and 14 as MCD. 10 renal biopsies from healthy living transplant donors were used as normal controls. Immunohistochemical stains with an antibody against Sox-9 were performed on representative slides from each case. Specimens with sampling of 5 or more glomeruli were considered adequate. Sox9 nuclear staining was recorded as positive/negative in glomeruli (mesangial cells/podocytes), and as percentage of stained cells in parietal and tubular epithelial cells (PEC/TEC).
Results: Ages ranged from 1-18 years, with 14 males and 15 females. 5 cases (3 FSGS and 2 MCD) were excluded due to sampling inadequacy. None (0/10) of the normal controls showed glomerular staining. 7/12 (58.3%) FSGS cases had positive glomerular staining (seen in segmentally sclerotic and non-sclerotic glomeruli) versus 1/12 (8.3%) MCD cases (p=0.0136). Sox9 stained 0-10% of TEC and PEC in all controls (100%) and most MCD (67%), and 10-50% in most FSGS cases (67%).
Conclusions: Glomerular Sox9 expression was significantly more frequent in FSGS than MCD, suggesting a potential use for this protein as a diagnostic/prognostic marker of fibrogenesis in NS. A larger patient sample is needed to confirm these observations, to establish a possible relationship between Sox9 staining and poor outcome in MCD, and an association between tubular Sox9 staining and chronic parenchymal changes.
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Mesangial Damage and Repair: The Role of Stem Cells. A 6D Living Cell Model.
Jiamin Teng, Elba A Turbat-Herrera, Guillermo A Herrera. Nephrocor, Tempe, AZ
Background: In light chain deposition disease and AL-amyloidosis, mesangial cells are injured by GLCs resulting in MC apoptosis. However, the changes that occur in the mesangium are diametrically different. In AL-amyloidosis, mesangiolysis occurs whie in light chain deposition disease, the matrix increased and changes its compostion. Repair mechanisms of the affected mesangium are poorly understood. Stem cells may play an important role in repairing the damaged mesangium, especially since not many mesangial cells typically remain after the damage.
Design: Rat MCs were cultured on Matrigel loaded glass bottomed multi-well plates with 10% FBS/RPMI 1640 until confluence. MCs were then made quiescent by incubating them with 0.5% FBS/RPMI for 48 hours and then treated with LCDD and AL-AmLCs (10 ug/ml) purified from the urine of patients with renal biopsy proven conditions. Rat mesenchyma stem cells (RMSCs) are then stained with PKH-2 fluorescence or with Lysotracker Texas Red and placed 96 hours later into the wells of GLCs treated MCs for 2 weeks. The entire process is carefully monitored with sequential photos every 15 minutes using a 6D living cell model. This allows to observe the process of mesangial damage and how stem cells repair the damage.
Results: MCs treated with LCDD-LCs produced large amounts of matrix and formed nodules and those treated with AL-Am LCs engaged in the production of amyloid. MSCs cleaned up apoptotic MCs and eventually replaced injured cells becoming the majority of the cell mass. The amount of matrix material and amyloid decreased proportionally to the number of MSCs replacing the original MCs.
Conclusions: The reported observations with the 6D living cell imaging system demonstrated that the GLC-induced alterations are not irreversible and highlight a crucial role for MSCs replacing injured MCs and re-establishing mesangial homeostasis. In light chain deposition disease the process is complicated by the need to initially completely break down the abnormal accumulated matrix. These results suggest a therapeutic role for MSCs in repairing glomerular damage.
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本帖最后由 于 2011-03-06 09:36:00 编辑
The Renal Distribution of Iron (Fe) in Primary Hemochromatosis: An Autopsy Study.
Vijayalakshmi Ananthanarayanan, Shane Meehan, Anthony Chang. University of Chicago, IL
Background:
The distribution of iron (Fe) within the renal parenchyma of patients with primary hemochromatosis has not been studied. We conducted this study to determine the spectrum of Fe distribution in autopsy kidneys in the setting of primary hemochromatosis. We also evaluated whether the presence of the H63D or C282Y mutations, the most common mutations in hereditary hemochromatosis, resulted in a difference in iron distribution in the kidneys of affected patients.
Design:
We identified 12 cases of hemochromatosis in our pathology database from 1993-2010, after excluding cases of hemosiderosis and secondary hemochromatosis. H&E and Prussian blue stain were analyzed and the distribution of Fe in the various compartments of the renal parenchyma was assessed. These findings were correlated with the available clinical and mutation data (H63D and C282Y).
Results:
Of the 12 cases of primary hemochromatosis, there were 8 males and 4 females with an average age of 54 years (range: 32 to 73 years). Eight patients had histologically proven cirrhosis while 4 patients had increased Fe deposition in the hepatocytes. In the kidneys, there typically was more staining for Fe in the cortex than the medulla. Within the cortex, Fe was noted in the glomeruli, proximal convoluted tubules (PCT) and distal nephron segments. In most cases, the staining in the distal nephron segments, when present, was more intense than the PCT. Podocytes and parietal epithelial cells were the primary cells in glomeruli with Fe, but no mesangial or glomerular endothelial staining was identified. One case showed the presence of Fe in only glomeruli limited to the podocytes. Few cases had additional rare interstitial and endothelial staining. Furthermore, patchy staining of the glomerular basement membranes in globally sclerotic glomeruli, Bowman's capsules and tubular basement membranes was also noted in a subset of cases. In the four cases where mutation data was available, no difference in Fe distribution was observed.
Conclusions:
This is the first study to describe the distribution of Fe in autopsy kidneys of primary hemochromatosis patients. Most cases showed a predominant staining pattern in the cortex with a greater distribution in the distal nephron segments. Regardless, of the involved compartment, Fe deposition was patchy and irregular. The persistence of iron deposition in the podocytes could be due to their terminal differentiation, but this would require further study.
