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- 2011 USCAP Meeting Abstracts (2)
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Segmental Collapsing Capillary Tufts (SCCT) in Diabetic Glomeruli.
L Clarke Stout. University of Texas Medical Branch, Galveston
Background: Capillary obliteration is obvious in large Kimmelstiel-Wilson nodules, but its mechanism is unknown in the far more prevalent diffuse lesion, where expanding mesangium just seems to fill the glomerulus until it becomes obsolescent. This report describes novel segmental collapsing capillary tufts (SCCT) in 2 of 25 diabetics, 1 with Type 1 (T1DM) and 1 with Type 2 (T2DM) diabetes.
Design: SCCT were found by chance in a study of glomerular changes in 74 diabetics (end stage renal disease excluded) and 59 matched controls from consecutive autopsies at our hospital. The present study group consisted of 25 of the diabetics that had any degree of diabetic glomerulopathy and 12 matched controls. All 37 cases had 18 variously stained paraffin embedded 4 µm serial sections. One hundred glomeruli from each case were reviewed in section 9 (PAS). Nine to 15 glomeruli were completely traced in each of 2 diabetics and 2 controls that had PolyBed 812 embedded toluidine blue stained 1µm serial sections.
Results: SCCT were first seen in 1 μm serial sections in the T1DM case that had 16 SCCT in 8 of 9 complete and 4 partial glomeruli (5 gomeruli had 1, 1 had 2, 1 had 3 and 1 had 6 SCCT). Six SCCT in 5 glomeruli were large. No definite SCCT were found in the 4 µm sections from the same case. SCCT were called early [mild decrease in capillary lumen size and glomerular capillary basement membrane (GBM) width], mid (moderately decreased lumen size and GBM width), and late (collapsed autolyzed masses of barely recognizable capillaries and mesangium). SCCT were called small (in-situ capillary segments) or large (detached capillaries and mesangium up to 35 x 60 x >55µm in size. Three large SCCT were attached to the tuft by a single capillary, the GBMs of which were contiguous but abruptly thinned in the SCCT. Mesangium occupied the intracapillary space in the tuft at the junction. One T2DM case had 1 probable and 1 possible large SCCT in paraffin sections.
Conclusions: The decreased intraluminal volume and thin GBMs of SCCT suggested decreased perfusion. This and their seemingly autolytic demise suggested a gradual rather than a sudden decrease in flow, possibly due to diabetic mesangial expansion and/or disordered mesangial contraction. Inter or intralobular small anastomotic capillary loops would seem likely candidates for SCCT. Their obliteration could account for the glomerular lobulation and simplification typically seen in advanced diabetic glomerulopathy. The frequency of SCCT in 1µm plastic sections and their absence in 4µm paraffin sections in the same case suggest that SCCT may be underrecognized.
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Cross-Species Identification of Conserved Glomerular
Transcriptional Networks of Progressive Diabetic Nephropathy in Mouse
and Man.
Jeffrey B Hodgin, Viji Nair, Hongyu Zhang, Ann Randolph, Raymond C Harris, Robert G Nelson, Frank C Brosius, Matthias Kretzler. University of Michigan, Ann Arbor; Vanderbilt University, Nashville, TN; NIDDK, Phoenix, AZ
Background: Mouse models of diabetic nephropathy (DN) are valuable tools, but no mouse model reliably exhibits all features of human disease, hindering our ability to identify specific factors that cause or predict DN. To define where mouse models of diabetic glomerulopathy (DG) faithfully recapitulate human DG on a functional level using a cross-species comparison to identify shared transcriptional networks.
Design: Transcriptional networks for diabetic humans and AMDCC mouse models were generated using glomerular mRNA, Affymetrix microarrays, transcriptional pathway mapping, and promoter modeling tools. The human transcriptional network was derived from albuminuric (>30 mg/g Alb/Cr) versus nonalbuminuric (<30 mg/g Alb/Cr) Pima Indians, a cohort with early, progressive DG, and from later stage diabetics versus living donor controls. Mouse transcriptional networks were derived from streptozotocin treated DBA/2 mice, db/db C57BLKS mice, and eNOS-deficient db/db C57BLKS mice, each versus control.
Results: Integrating the gene expression alterations with biological knowledge resulted in complex networks of 1000s of genes linked by multiple co-citations and promoter binding sites (Genomatix Bibliosphere). TALE (Tool for Approximate Large Graph Matching, University of Michigan) was used to align the human and mouse transcriptional networks to derive three conserved network structures for each human-mouse comparison, comprised of approximately 100 nodes representing key hubs of conserved regulatory events. Shared gene nodes were found in all three networks, many of them reflecting established pathogenetic mechanisms of diabetic complications including JAK-STAT, VEGF, c-kit, and HGF-R signaling pathways. Shared top biological processes included cytokine mediated signaling, response to steroid hormone, and JAK-STAT cascade.
Conclusions: Comparing TALE networks from early progressive DG (PIMA albuminuric versus nonalbuminuric) against established DG (albuminuric diabetics versus nondiabetics) revealed pathways specific to glomerular disease progression. This approach can guide the selection of disease pathways in mouse models that are the most relevant to the human disease process and identify new pathways that are excellent targets for future study.
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Membranous Glomerulonephritis with Crescents.
Erika F Rodriguez, Samih H Nasr, Sanjeev Sethi, Mary E Fidler, Lynn D Cornell. Mayo Clinic, Rochester, MN
Background: Only rare cases of membranous glomerulonephritis (MGN) with crescents have been reported. We present a series of ANCA-negative MGN with crescents.
Design: We searched the pathology database at our institution from 1/1994-9/2010 for cases of MGN with crescents from non-lupus patients who had negative ANCA and anti-GBM antibodies. We examined in detail the clinicopathologic features and outcomes in these patients.
Results: A total of 4215 cases of MGN, both primary and secondary, were identified. Of these, 12 patients (0.3%) had ANCA- and anti-GBM-negative crescentic MGN. The mean age at diagnosis was 47 yrs (range 5-86); half were female. At presentation, all patients had proteinuria (mean 11.3 g/d; range 3.3-26) and hematuria, and 10/12 (83%) patients had renal failure (mean serum Cr 3.3 mg/dL; range 0.4-10). ANA was negative in 9 patients and transiently or weakly positive in 3 patients; no patients had hepatitis B or C infection. On light microscopy, glomeruli showed on average 26% involvement by crescents (range 5-73%), with 10/12 cases showing <50% crescents. All cases showed a membranous pattern; 6 cases showed mild mesangial and 2 showed segmental endocapillary proliferation. By immunofluorescence, all cases with glomeruli (n=11) showed a membranous pattern with staining for IgG, kappa, and lambda, and all but one showed C3; 2 cases showed staining for C1q (trace and 2+) and one showed trace IgA. By electron microscopy, most cases showed stage II MGN. 2 showed mesangial deposits, and one showed subendothelial deposits. Endothelial tubuloreticular inclusions were seen in one case. Follow-up clinical data were available in all 12 cases (mean 32 mos; range 0.5-138). 10 patients were treated, 5 with high-dose prednisone and cyclophosphamide, 2 with steroids and a calcineurin inhibitor (tacrolimus or cyclosporine), 2 with prednisone alone, and one with enalapril alone. 2 patients progressed to ESRD, at 0-2 mos post-biopsy. The mean serum Cr of the other 10 patients at follow-up was 1.6 mg/dL (range 0.5-4.1), with 8/10 (80%) patients showing elevated serum Cr. 6 of 7 patients with available data and without ESRD had ≥1 g/d proteinuria at follow-up. One patient later had an equivocal MPO titer, and another had a positive MPO titer 8 mos after biopsy. No patients developed lupus. 2 patients had follow-up biopsies, at 1.3 and 4.6 years later: one had MGN without crescents, and the other had MGN with focal crescents.
Conclusions: Crescentic MGN is a rare variant of MGN that usually presents with heavy proteinuria, hematuria, and renal failure. The prognosis is variable and the disease may respond to therapy, but most patients develop chronic renal failure.
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Expansion of the Parietal Cell Compartment in Collapsing Glomerulopathy.
Miguel F Palma Diaz, Giovanna Giannico, Sharon Phillips, Yu Shyr, Agnes B Fogo, Charles E Alpers, Vivette D D'Agati. Columbia University, New York, NY; University of Washington, Seattle; Vanderbilt University, Nashville, TN
Background: Glomerular epithelial cell hyperplasia and pseudocrescent formation overlying collapsed segments are defining features of collapsing glomerulopathy. The proliferative potential and relative contributions of dedifferentiated podocytes (PODs) versus parietal epithelial cells (PECs) to the glomerular epithelial hyperplasia remain controversial.
Design: We examined 44 biopsies of primary collapsing focal segmental glomerulosclerosis classified by the Columbia classification. Serial paraffin sections were stained with antisera to claudin (an occludens junction protein expressed on PECs and tubular cells), WT-1 (a zinc finger transcription factor expressed by mature PODs) and Ki-67 (proliferation marker). Every glomerulus (including collapsing, NOS and histologically normal phenotypes) in each biopsy was scored for positive cells in lesional vs non-lesional segments and dichotomized for visceral and parietal anatomic locations. Co-localization was investigated by superimposition of serial images.
Results: Histologically normal glomeruli displayed restriction of claudin to PECs lining Bowman's capsule, whereas WT-1 was expressed by visceral PODs and rare parietal PODs, suggesting non-overlapping lineage-specific markers. Variable numbers of claudin+, Ki67+ cells could be identified lining Bowman's capsule in collapsing, NOS and histologically normal glomeruli. There was widespread downregulation or complete loss of WT-1 overlying collapsed segments with swollen PODs. Only rare PODs bearing WT-1 were also Ki-67+. By contrast, most Ki-67+ glomerular epithelial cells were strongly claudin+, indicating predominant expansion of the parietal compartment. Claudin+ cells typically extended out from Bowman's capsule to bridge and overlie the glomerular tuft in acute collapse and more chronic NOS lesions, often mimicking PODs, and formed the majority of cells in the pseudocrescents.
Conclusions: Loss of WT-1 from PODs in collapsing glomerulopathy supports the concept of the dysregulated podocyte phenotype in the pathogenesis of this lesion. PECs can populate the collapsed tuft and comprise the majority of cell cycle-engaged cells in primary collapsing glomerulopathy.
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Angiotensin II Type I Receptor Blocker Is Limited in Protection Against Severe Podocyte Injury-Induced FSGS.
Hai-Chun Yang, Shu-Wei Wang, Ira Pastan, Taiji Matsusaka, Iekuni Ichikawa, Agnes B Fogo. Vanderbilt University Medical Center, Nashville, TN; National Cancer Instiute, National Instiute of Health, Bethesda, MD; Tokai University Medical School, Isehara, Kanagawa, Japan
Background: In 5/6 Nx models in both rats and mice, we found high dose angiotensin II type I receptor blocker (ARB) prevented glomerulosclerosis progression and even could induce regression. Nep25 mice, which express the human CD25 receptor on podocytes, can dose-dependently develop progressive glomerulosclerosis when immunotoxin (LMB2) is administered and binds this receptor. Previously we have shown that renal collagen I expression was parallel to luciferin imaging in double transgenic Nep25/collagen I-luciferase mice with glomerulosclerosis induced by this toxin. In this study, we aimed to examine if ARB could protect in this model of severe primary podocyte injury-associated FSGS.
Design: Collagen I-luciferase/Nep 25 mice were exposed to a mean dose of LMB2 toxin (12.5 ng/g BW, i.p.). Mice were biopsied at week 2, randomly assigned to the ARB losartan (200mg/L) or not treated (control group), then sacrificed at week 6. Podocyte number, urine total protein/creatinine ratio, glomerulosclerosis index (SI, 0-4 scale) and collagen I associated luciferase activity were measured.
Results: LMB2 reduced podocyte number about 40% assessed by WT-1 staining (normal 7.50±0.50 vs. LMB2 4.64±0.11 /glomerulus). The control group showed edema and persistent proteinuria (week 2 66.06±14.51, week 4 72.52±11.40, week 6 87.33±29.57). Progressive glomerulosclerosis (SI, week 2 1.26±0.11 vs. week 6 1.81±0.04, p<0.05) and increased collagen I activity also developed (luciferase imaging score, week 2 0.84±0.13, week 4 1.66±0.09, week 6 1.79±0.001 ×e6, p<0.05 overtime) in control toxin-treated mice. Losartan stabilized collagen I activity (luciferase imaging score, week 2 0.96±0.18, week 4 0.78±0.10, week 6 1.06±0.13 ×e6). However, ARB did not decrease proteinuria (week 2 96.18±16.53, week 4 94.10±11.96, week 6 73.76±7.92, pNS) and did not prevent glomerulosclerosis progression (SI, week 2 1.48±0.15 vs. week 6 2.09±0.19, p<0.05).
Conclusions: Our data indicate that severe podocyte loss causes progressive glomerulosclerosis, and ARB alone cannot prevent glomerulosclerosis progression in this setting. This may be related to continuing podocyte loss cycle which accelerates the progression process.
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Application of the Columbia Classification of Focal Segmental
Glomerulosclerosis in Kidney Biopsies from Patients with HIV Infection.
Shane M Meehan, Lisa Kim, Anthony Chang. University of Chicago, IL
Background: The Columbia system of classification of focal segmental glomerulosclerosis (FSGS) identifies 5 different types or variants of this distinct glomerular lesion. FSGS of the collapsing variant is a classical feature of HIV associated nephropathy. Other variants of FSGS have also been described in this disorder. We examined the spectrum of FSGS lesions in biopsies obtained from patients with HIV infection using the Columbia classification system.
Design: We identified 43 renal biopsies from 42 patients with HIV infection obtained over a 7 year period. Twenty five biopsies had FSGS. Reproducibility of the Columbia system was good (kappa 0.65).
Results: Sixteen biopsies had FSGS, collapsing variant (coll) (64%). Six had FSGS not otherwise specified (NOS) (24%). Fifteen biopsies (60%) had focal glomerular capillary collapse without visceral epithelial hyperplasia (10 coll, 4 NOS, 1 cell). One each had cellular (cell), tip lesion (T) and perihilar (Ph) variants. Glomeruli with features of multiple variants of FSGS were frequently identified in the same biopsy and included coll with NOS (n=9), cell (n=5), or Ph (n=3), cell and NOS (n=1), T and NOS (n=1).
Conclusions: It is no surprise that collapsing FSGS was the commonest variant observed in biopsies from HIV infected patients. NOS lesions were less common and other variants rare. Mixed Columbia FSGS lesions were evident in more than half of this sample (52%). Mixed patterns of FSGS raise the possibility that Columbia FSGS variant lesions may form part of a spectrum of glomerular responses to injury in HIV infection.
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Urinary Polyomavirus Haufen Shedding Accurately Reflects
Intrarenal Burden of Polyomavirus Nephropathy (PVN): Comparative
Quantitative Analyses of Different Screening Techniques.
Harsharan Singh, Kozlowski Tomasz, True Karen, Vimal Derebail, Detwiler Randy, Adil Gasim, Volker Nickeleit. The University of North Carolina, Chapel Hill
Background: We previously defined a new, qualitative noninvasive test to accurately diagnose PVN, a common infectious complication in renal allografts. The test is based on the detection of three dimensional cast-like polyomavirus (PV) aggregates, "Haufen", in voided urine samples. Urinary Haufen-testing is highly accurate for PVN (positive and negative predictive values > 95%).
Aim: Quantitative assessment of urinary Haufen shedding, viremia and viruria (by PCR, urine cytology) was performed and correlated with intrarenal signs of PV replication/PVN (viral detection by immunohistochemistry: SV40-T antigen). Hypothesis: In comparison to conventional screening techniques, the degree of urinary Haufen shedding most accurately reflects the extent of intrarenal PV replication and "viral burden".
Design: Quantitative analysis of urinary Haufen shedding (number Haufen/mL), PCR assays (serum, urine), and quantitation of urinary decoy cell shedding (number decoy cells/ThinPrep® slide) were performed on 39 samples from 37 patients with concurrent biopsy proven PVN. Results were correlated with biopsy findings (PVN disease stage and number of cells / %tubules expressing SV40-T antigen). Statistics were performed using Kruskal Wallis testing with ties.
Results: Degree of intrarenal polyomavirus replication:PVN disease stages: Only the number of urinary Haufen/mL showed a significant correlation with PVN disease stages (p<0.0001). Neither the degree of viremia nor viruria and decoy cell shedding correlated with PVN disease stages.
Conclusions: Haufen-testing is the most accurate noninvasive technique to diagnose PVN, predict PVN disease stages, and also to monitor the degree of polyomavirus replication. It may help to better assess the effectiveness of anti-viral therapy during persistent disease The number of urinary Haufen/mL showed the tightest correlation with the number of SV40-T expressing cells (correlation coefficient (cc) 0.86) and with the percentage of affected tubules (cc 0.70) in allografts with PVN (in comparison: cc viremia by PCR: 0.45; cc viruria by PCR: 0.45; cc decoy cells: 0.01). .
