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- 2011 USCAP Meeting Abstracts (5)
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Microcirculation Endothelial Cell Proliferation Is Specifically
Increased in Antibody-Mediated Rejection as Compared to Other Diseases
in Kidney Transplants.
Stephen Osasan, Jessica Chang, Yasemin Ozluk, Declan G de Freitas, Michael Mengel, Phillip Halloran, Banu Sis. University of Alberta, Edmonton, Canada
Background: Antibody-mediated rejection (ABMR) is mediated by antibodies against donor HLA, leading to allograft injury and failure. We hypothesized that there is greater endothelial repair response in ABMR compared to other diseases in kidney transplants.
Design: We related microcirculation endothelial cell cycling and density of peritubular capillaries(PTC) to lesions, diagnoses, and whole-genome microarrays in 100 non-selected kidney transplant biopsies for cause(BFC) and 20 normal kidney implantation biopsies from living donors. We performed double immunostaining and quantified the microcirculation endothelial cell cycling (Ki-67+CD31+ glomerular capillaries and PTCs) and the CD31+ PTC density by cell counting in the entire renal cortical biopsy area.
Results: The BFC showed higher numbers of cycling microcirculation endothelial cells in comparison to normal controls (p=0.001). The endothelial cell cycling was specifically increased in ABMR compared to other diseases i.e. T cell-mediated rejection, glomerulonephritis, and others.
This increase correlates with microcirculation lesions (glomerulitis, peritubular capillaritis, transplant glomerulopathy), and C4d staining (p<0.05). Furthermore, It also correlates with transcript sets representing the molecular burden of active ABMR(p<0.05). The PTC density was lower in biopsies for cause when compared to normal controls (p<0.001). However, the PTC density did not differ among diagnoses and was not correlated with time post-transplant, scarring or other lesions.
Conclusions: We conclude that endothelial repair response is specifically increased in kidneys with ABMR, reflecting the burden of microcirculation injury. In contrast to previous observations, there is no evidence for reduced PTC density in kidneys with scarring.
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C4d/CD34 Co-Immunofluorescence Staining for the Evaluation of the Extent of C4d Positivity in Renal Transplants.
Kuang-Yu Jen, Thuy B Nguyen, Zoltan G Laszik. University of California San Francisco
Background: Immunofluorescence (IF) detection of the complement split product C4d along tubulointerstitial (TIS) capillaries of transplant kidney biopsies is the mainstay of diagnosing antibody-mediated rejection (AMR). The extent of C4d positivity may have significant clinical ramifications; however, precise quantitative assessment of the proportion of TIS capillaries that are C4d-positive is often difficult, if not impossible. The aim of our study is to develop a C4d/CD34 double IF stain that allows not only rapid and sensitive detection of C4d positivity, but also precise and reproducible morphometric quantitation of the fraction of C4d-positive TIS capillaries.
Design: Renal transplant biopsies with C4d-positive acute or chronic AMR (n=10) as determined by single IF staining for C4d on frozen sections were used for the study. Two biopsies negative for C4d IF were used as negative controls. Frozen sections were stained with a mixture of polyclonal rabbit anti-C4d (ALPCO) and mouse anti-CD34 (Dako) antibodies followed by incubation with a mixture of fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit (Vector) and Texas Red-labeled horse anti-mouse (Vector) antibodies. Digital photographs of multiple fields were taken from each double-stained slide at 20x magnification for FITC and Texas Red IF. The photographs were processed using Just Another Colocalization Plugin (JACoP) for the ImageJ® software in order to calculate the extent of colocalization of C4d and CD34 positivity in the TIS capillaries. Manders coefficients M1 and M2 were used to determine the extent of colocalized staining. To assess the sensitivity of the C4d/CD34 double IF stain for C4d detection, the findings of C4d positivity in the double-stained sections were visually compared to the corresponding fields of serially-cut frozen sections stained for C4d alone.
Results: The extent and intensity of the C4d positivity was comparable in the double (C4d/CD34) and single (C4d) IF-stained sections. Colocalization of C4d and CD34 in the TIS capillaries as assessed by Manders coefficient M2 ranged from 0.17 to 0.87. A high degree of correlation was observed between various fields in the same biopsy for the extent of colocalization of C4d and CD34 positivity.
Conclusions: C4d/CD34 double IF stain on frozen sections yields rapid and sensitive detection of TIS capillary C4d positivity in renal transplant biopsies. It also allows simple, precise, and reproducible morphometric determination of the C4d-positive fraction of the CD34-positive TIS capillaries.
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Deficiency of B Cells but Not Mast Cells Ameliorates Chronic Allograft Damage in a Mouse Model for Renal Transplantation.
Michael Mengel, Konrad Famulski, Jessica Chan, Luis Hidalgo, Banu Sis, Philip Halloran. University of Alberta, Edmonton, Canada
Background: We recently observed that the number of B cells and mast cells increases with time post transplantation in human renal allografts. Furthermore these two cell types predominately accumulate in areas of interstitial fibrosis and tubular atrophy (IFTA), suggesting a potential pathogenetic role of these cell in the onset and/or progression of IFTA.
Design: We analyzed the long-term time course of transplanting a wild type allograft (CBA) into a B cell (B6.129S2-Igh-6-/-) or mast cell (C57BL/6-KitW-v/W-v) deficient recipient and compared the findings to respective controls (CBA into B6 wild type). This strain combination (CBA into B6) represents transplantation across complete MHC incompatibility. Transplants were done in a non-life supporting model without any treatment and thus developed the natural course of rejection. Transplanted kidneys were harvested on day 7, 14, 21, 42, and 60 (total n=150) and analyzed by histopathology according to Banff criteria.
Results: B cell deficient recipients: At early time points (day 7 and 14) no significant differences in terms of interstitial infiltrates were found between controls and deficient recipients. On day 7, B cell deficient recipients showed significantly more tubulitis (p=0.004), but on day 21 a trend towards less interstitial inflammation and tubulitis. From day 42 and persisting to day 60, B cell deficient recipients showed significantly less, only focally present interstitial infiltrates and tubulitis. This resulted in significantly less interstitial fibrosis on day 60.
Mast cell deficient recipients: On day 7 and day 14 the deficient recipients showed significantly more interstitial fibrosis compared to controls. More mast cell deficient mice showed extensive necrosis and arterial thrombosis at late time points of harvest, i.e. day 42 and day 60.
Conclusions: Absence of B cells in the recipient has no influence on early cellular rejection of mice renal allografts. In contrast long-term maintenance of the inflammatory response and thus onset of chronic injury is dependent on the presence of B cells. In contrast mast cell deficient recipients show more vigorous acute rejection, potentially indicating an immune-modulatory role for this cell type.
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The Molecular Phenotype of Six-Week Protocol Biopsies from Human
Renal Allografts: Reflections of Prior Injury but Not Future Course.
Michael Mengel, Jessica Chang, Daniel Kayser, Wilfried Gwinner, Anke Schwarz, Gunilla Einecke, Verena Broecker, Konrad Famulski, Declan De Freitas, Luis Hidalgo, Hermann Haller, Banu Sis, Philip Halloran. University of Alberta, Edmonton, Canada; Hannover Medical School, Germany
Background: Protocol biopsies after renal transplantation are considered a surveillance tool for detection of subclinical pathologies at a stage where they are potentially more amenable to therapy.
Design: We assessed the molecular phenotype of 107 six-week protocol biopsies from human renal allografts, using Affymetrix microarrays. Transcript changes were summarized as pathogenesis-based transcript sets (PBTs) reflecting inflammation (T cells, macrophages, IFNG effects) and the injury-repair response of the parenchyma, stroma, and microcirculation – increased (“injury-up”) and decreased (“injury-down”) transcripts.
Results: The molecular changes were highly correlated with each other, even when all rejection and borderline cases were excluded. Inflammation and injury-down PBTs correlated with histologic inflammation and tubulitis, and the inflammation transcripts were greater in kidneys diagnosed as T cell-mediated or borderline rejection. In terms of their molecular phenotype no difference between borderline and T cell mediated rejection was found (figure 1a). Injury-up PBTs did not correlate with histopathology but did correlate with kidney function: thus functional disturbances are represented in transcript changes but not in histopathology. PBT changes correlated with prior delayed graft function. However, there was little difference between live donor kidneys and deceased donor kidneys that had not shown delayed graft function. Molecular changes did not predict future biopsies for clinical indications, rejection episodes, functional deterioration, or allograft loss. Despite the fact that cases with T cell mediated rejection (n=9) were treated and those with borderline (n=20) not, and both showed a similar molecular phenotype, no difference in terms of outcome between these two Banff groups were observed (figure 1b).
Conclusions: Thus while detecting T cell-mediated inflammation, the molecular phenotype of early protocol biopsies mostly reflects the injury-repair response to implantation stresses, and has little relationship to future events and outcomes, independent of any therapeutic intervention.
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Green Tea Polyphenol (-)-Epigallocatechin-3-Gallate (EGCG) Ameliorates Experimental Immune-Mediated Glomerulonephritis.
Ting Ye, Ai Peng, Dinesh Rakheja, Chandra Mohan, Xin J Zhou. UT Southwestern Medical Center, Dallas, TX
Background: Oxidative stress and inflammation play critical roles in the pathogenesis of immune-mediated glomerulonephritis (GN). The green tea catechins, particularly (-)-epigallocatechin-3-gallate (EGCG), are potent anti-inflammatory and anti-oxidant agents shown to inhibit leukocyte chemotaxis, quench free radicals, chelate transition metals, and interrupt lipid peroxidation chain reaction. It is estimated that EGCG is 25 times more potent than vitamin E and 100 times more potent than vitamin C making it an attractive compound for the treatment of diseases such as anti-GBM-GN that are characterized by severe immunologic and oxidative injury. However, the effect of EGCG on the clinical and pathological progression of immune-mediated renal injury has not been investigated.
Design: We tested the hypothesis that the anti-inflammatory and anti-oxidant properties of EGCG may favorably affect the course of immune-mediated GN using a murine model of anti-GBM-GN. 129/svJ mice were challenged with rabbit anti-mouse-GBM sera to induce GN and subsequently divided into EGCG (50 mg/kg/day, orally) and vehicle treated groups. Routine histology, markers of oxidative stress and key molecules associated with redox and inflammatory pathways were studied.
Results: Vehicle-treated mice developed glomerular crescents, tubulointerstitial disease and inflammatory cell infiltrates. EGCG treatment led to reduced proteinuria and serum creatinine and marked improvement in renal histology. Vehicle-treated mice showed increased oxidative stress [malondialdehyde (MDA), H2O2 and nitrotyrosine], elevated osteopontin (OPN), p65-NFκB, inducible nitric oxide synthase (iNOS), NO metabolites, superoxide dismutase (SOD), myeloperoxidase (MPO), p-Akt, and p47phox, and reduced PPARγ, glutathione peroxidase (GPx), and catalase activity. Treatment with EGCG led to a reduction in oxidative stress, normalization of OPN, p65-NFκB, iNOS, NO metabolites, p-Akt, p47phox, MPO, GPx, and PPARγ, and a significant increase in SOD expression.
Conclusions: our data suggest that EGCG ameliorates laboratory and histologic abnormalities in the mouse model of immune-mediated GN. These salutary effects of EGCG are likely mediated by the anti-inflammatory and anti-oxidative properties of this compound. These observations demonstrate the potential utility of EGCG as a novel therapeutic agent for the treatment of immune-mediated glomerulonephritides and various other immune-mediated diseases。
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Arteriolar Sclerosis in Tumor Nephrectomy Specimens Is Prognostically Significant.
Steven P Salvatore, Sruthi N Reddy, Eugene K Cha, James S Rosoff, Surya V Seshan. Weill Cornell Medical College, New York
Background: Recent studies have shown that evaluating non-tumor portions of tumor nephrectomy specimens is useful in diagnosing non-neoplastic renal disease. The purpose of this study is to determine the frequency of medical renal disease and to assess the prognostic significance of the degree of vascular sclerosis in the long term follow-up of tumor nephrectomy patients.
Design: We reviewed non-neoplastic kidney H&E stained slides of 232 cases from 1998 to 2005. Sixteen cases were excluded from the study (6 tumor compression, 5 no uninvolved kidney, 5 embolization/ infarction). PAS staining, immunofluorescence, and/or electron microscopy was performed where appropriate. Vascular sclerosis was scored from 0-3, mild, moderate and severe. Follow-up of at least 1 year was evaluated in 101 cases. The degree of vascular sclerosis was compared to the change in the creatinine level, pre-surgery to follow-up (Table 1).
Results: Of the 216 cases reviewed, 47 had new pathologic diagnoses (21%): diabetic nephropathy 21, hypertensive nephropathy 11, focal segmental glomerulosclerosis 6, collapsing glomerulopathy 2, acute pyelonephritis, thrombotic microangiopathy, atheroembolic disease, granulomatous interstitial nephritis, reflux nephropathy, proliferative glomerulonephritis, and membranous glomerulonephritis 1 each. Eighty percent (80%) of the cases with additional non-neoplastic diagnoses and follow-up showed severe arteriolar sclerosis. Global glomerulosclerosis (GS) was higher in cases with more severe vascular sclerosis, mean 4.5% GS (mild) versus 17% GS (severe). Three patients, all diabetic nephropathy, progressed to end stage renal disease from 1 to 4 years after nephrectomy.
| Arterioles | N | mean Cr difference (mg/dL) | p value | mean f/u (yrs) |
| mild | 42 | 0.3 | 0.006* | 5.6 |
| moderate | 33 | 0.4 | 0.02* | 5.1 |
| severe | 26 | 0.8 | n/a | 4.9 |
| Arteries | ||||
| mild | 47 | 0.3 | 0.09 | 5.9 |
| moderate | 33 | 0.5 | 0.23 | 4.2 |
| severe | 21 | 0.7 | n/a | 4.4 |
Conclusions: Non-neoplastic renal pathology can be evaluated in the majority of tumor nephrectomy specimens with 21% having additional diagnoses. The degree of arteriolar sclerosis but not arteriosclerosis is predictive of significantly higher creatinine levels and subsequent decline in renal function at follow-up in post-nephrectomy patients. The prognostic implications of the non-tumor pathology are therefore important in patients with reduced renal mass.
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Hypertension-Associated Nephrosclerosis: Does the Activation of
Parietal Epithelial Cells (PECs) Differ between African Americans and
Caucasians?
M Olatokunboh Odubanjo, Bart Smeets, Marcus J Moeller, Agnes B Fogo. Vanderbilt University Medical Center, Nashville, TN; University Hospital of RWTH, Aachen University, Germany
Background: Loss of podocytes is critical for progressive glomerulosclerosis. Parietal epithelial cells (PECs) may act as stem cells that replenish podocytes. Our aim was to determine the extent to which PECs are activated following hypertension-associated injury in African Americans (AA) and Caucasians (C).
Design: Activated PECs were identified by CD44 staining. We scored activated PECs in anatomical parietal and podocyte locations in glomeruli without lesions and glomeruli with segmental sclerosis (SS) and/or adhesions, and the total number of activated PECs was quantified for globally sclerosed (GS) glomeruli.
Results: A total of 352 glomeruli from 31 biopsies were analyzed. 209 glomeruli showed no lesions (50 AA, 159 C), 110 (73 AA, 37 C) showed global sclerosis, 12 (9 AA, 3 C) showed SS and/or adhesions, 19 (10 AA, 9 C) showed periglomerular fibrosis, and 2 (both AA) showed extracapillary proliferation with no GBM breaks or fibrinoid necrosis. The distribution of glomerular lesions differed between AA and C, with glomeruli without lesions being more commonly seen in C, and GS and SS being more common in AA.
Activated PECs were detected in 21 glomeruli, 14 AA and 7 C (8 glomeruli without lesions: 5 AA and 3 C; 4 with GS: 3 AA and 1 C; 4 with SS and or adhesions: all AA; 3 with periglomerular fibrosis: all C; and 2 with extracapillary proliferation: both AA).
There was an average of 7.2 CD44+ cells in parietal location/ glomerulus in AA versus 1.7/ glomerulus in C.
In 4 of the 12 glomeruli with SS and/or adhesions, CD44+ cells were most common in a parietal location and more commonly, away from the lesion than adjacent to the lesion (4 glomeruli, on average 7.25/glomerulus, 6 in parietal location (1 in the lesion and 5 in the non-lesion area) and 1.25 in podocyte location (P value < 0.001 for lesion versus non-lesion area).
Conclusions: Activated PECs were present more commonly in AA than C, even in glomeruli without sclerosing lesions. This supports the possibility that abnormalities in the differentiation and regulation of the glomerular epithelial cell population could contribute to the pathogenesis of the glomerular scarring in arterionephrosclerosis. Whether these activated PECs contribute to sclerosis or represent regenerative repair responses awaits further study.
