Glomerular Basement Membrane Duplication Research Articles

Pathological Analysis of Early Transplant Glomerulopathy in Renal Allografts Using Low-Vacuum Scanning Electron Microscopy

Aim: Low-vacuum scanning electron microscopy (LVSEM) has been reported to aid in diagnosis of renal biopsy. This study evaluated early transplant glomerulopathy in kidney transplant recipients using LVSEM. Methods: We selected 4 biopsies of cg0, 5 biopsies of cg1a, 5 biopsies of cg1b, and 4 biopsies of cg2 lesions that had been evaluated by light microscopy (LM) and transmission electron microscopy from recipients with acute/active or chronic, active antibody-mediated rejection (AABMR or CAABMR). Renal allograft paraffin sections (1 µm thickness) were stained with periodic acid-methenamine silver and observed using LVSEM. The cg score was based on the Banff classification. The parameter “percentage of duplicated capillary number” was calculated as follows: in 1 glomerulus with glomerular basement membrane (GBM) duplication, the total duplicated capillary number/the total number of capillaries ×100. Results: In all 4 biopsy specimens with AABMR showing cg0, LVSEM revealed GBM duplication not identified by LM. The average percentage of duplicated capillary number per glomerulus with GBM duplication was higher when observed by LVSEM than when observed by LM in all cg1b and cg2 biopsy specimens. Conclusion: LVSEM revealed early GBM duplication in AABMR. Early GBM duplication might progress in the very early phase of AABMR. GBM duplication was more frequently detected by LVSEM than by LM in biopsy specimens with early chronic, active antibody mediated rejection. Thus, LVSEM may be useful in diagnosis of early transplant glomerulopathy.

Early identification of transplant glomerulopathy in pediatric kidney transplant biopsies: A single-center experience with electron microscopy analysis.

Banff 2013 criteria recommend performing ultrastructural studies with electron microscopy (EM) in kidney transplant biopsies if the technology is available. We sought to determine the impact of EM on enhancing diagnostic findings in pediatric kidney transplant biopsies and the prognostic information gained from the additional findings. All kidney transplant biopsies since routine EM use started on June 1, 2014, until October 31, 2016, were reviewed. Primary outcome measures included the positive yield frequency of EM use defined as an upgraded diagnosis based on EM findings relative to light microscopy, and 12-month kidney allograft outcome of progression to ESRD or doubling of serum creatinine stratified by transplant glomerulopathy (TG) status on EM. Eighty unique kidney transplant biopsies were reviewed. EM studies were completed for 61 biopsies (76%). Complication rate was low (3.7%). In 61 biopsies where EM was completed, EM findings included foot process fusion (62%), endothelial cell swelling (38%), subendothelial lucencies (31%), and glomerular basement membrane duplication (41%). EM confirmed FSGS recurrence in three cases. In the remaining 58 cases, there was a positive yield of 31% where 18 biopsies were upgraded to a worse category after TG identification on EM. Kidney allograft outcome was poor regardless whether TG was detected early on EM or advanced on LM. Routine EM use in analyzing pediatric kidney transplant biopsies proved safe and provided valuable additional diagnostic information in almost one-third of cases. Additional studies are needed to determine if clinical interventions for early TG identified on EM can improve long-term outcomes.

C5b9 Deposition in Glomerular Capillaries Is Associated With Poor Kidney Allograft Survival in Antibody-Mediated Rejection.

C4d deposition in peritubular capillaries (PTC) reflects complement activation in antibody-mediated rejection (ABMR) of kidney allograft. However, its association with allograft survival is controversial. We hypothesized that capillary deposition of C5b9—indicative of complement-mediated injury—is a severity marker of ABMR. This pilot study aimed to determine the frequency, location and prognostic impact of these deposits in ABMR. We retrospectively selected patients diagnosed with ABMR in two French transplantation centers from January 2005 to December 2014 and performed C4d and C5b9 staining by immunohistochemistry. Fifty-four patients were included. Median follow-up was 52.5 (34.25–73.5) months. Thirteen patients (24%) had C5b9 deposits along glomerular capillaries (GC). Among these, seven (54%) had a global and diffuse staining pattern. Twelve of the C5b9+ patients also had deposition of C4d in GC and PTC. C4d deposits along GC and PTC were not associated with death-censored allograft survival (p = 0.42 and 0.69, respectively). However, death-censored allograft survival was significantly lower in patients with global and diffuse deposition of C5b9 in GC than those with a segmental pattern or no deposition (median survival after ABMR diagnosis, 6 months, 40.5 months and 44 months, respectively; p = 0.015). Double contour of glomerular basement membrane was diagnosed earlier after transplantation in C5b9+ ABMR than in C5b9– ABMR (median time after transplantation, 28 vs. 85 months; p = 0.058). In conclusion, we identified a new pattern of C5b9+ ABMR, associated with early onset of glomerular basement membrane duplication and poor allograft survival. Complement inhibitors might be a therapeutic option for this subgroup of patients.

Glomerular basement membrane duplication is a predictor of the prognosis of diabetic nephropathy in patients with type 2 diabetes.

Several clinical parameters and pathological findings are known to be predictors of the deterioration of diabetic nephropathy (DN). Glomerular basement membrane duplication (GBM-DP) is a pathological feature representing endothelial injury, which is commonly observed in DN. In the present study, we investigated the association between GBM-DP and the renal prognosis in DN. The study enrolled 80 patients with renal biopsy-proven DN who were managed at Chiba-East Hospital from 2005 to 2012. We confirmed the pathological findings according to the Renal Pathology Society classifications, and we further evaluated the GBM-DP, which was defined as double contours of the GBM that expanded more than 10% of capillary loops in the most affected nonsclerotic glomerulus. We used Cox regression models to estimate hazard ratios (HRs) for end-stage renal disease (ESRD), with adjustment for age, sex, systolic blood pressure, HbA1c, estimated glomerular filtration rate (eGFR), and urinary protein excretion (UP) at baseline. Of the 80 patients, 56 were male (70.0%) and the mean age was 59.1years. The median eGFR and UP were 42ml/min/1.73m2 (IQR 30, 59) and 3.1g/gCr (IQR 1.2, 5.2). Twenty-seven patients progressed to ESRD and one patient died during the median observational period of 2.9years (IQR 1.5, 4.3). The multivariable analyses showed that GBM-DP was significantly associated with ESRD (HR 3.18 [95% confidence interval (CI): 1.02-9.87], p = 0.045). We newly identified GBM-DP as a strong prognostic predictor in DN patients. Further study is needed to clarify the pathogenic mechanism of GBM-DP in DN.

Antibody-Mediated Rejection: A Review.

Chronic antibody injury is a serious threat to allograft outcomes and is therefore the center of active research. In the continuum of allograft rejection, the development of antibodies plays a critical role. In recent years, an increased recognition of molecular and histologic changes has provided a better understanding of antibody-mediated rejection (AMR), as well as potential therapeutic interventions. However, several pathways are still unknown, which accounts for the lack of efficacy of some of the currently available agents that are used to treat rejection. We review the current diagnostic criteria for AMR; AMR paradigms; and desensitization, treatment, and prevention strategies. Chronic antibody-mediated endothelial injury results in transplant glomerulopathy, manifested as glomerular basement membrane duplication, double contouring, or splitting. Clinical manifestations of AMR include proteinuria and a rise in serum creatinine. Current strategies for the treatment of AMR include antibody depletion with plasmapheresis (PLEX), immunoadsorption (IA), immunomodulation with intravenous immunoglobulin (IVIG), and T cell- or B cell-depleting agents. Some treatment benefits have been found in using PLEX and IA, and some small nonrandomized trials have identified some benefits in using rituximab and the proteasome inhibitor-based therapy bortezomib. More recent histologic follow-ups of patients treated with bortezomib have not shown significant benefits in terms of allograft outcomes. Furthermore, no specific treatment approaches have been approved by the US Food and Drug Administration. Other agents used for more difficult rejections include bortezomib and eculizumab (an anti-C5 monoclonal antibody). AMR is a fascinating field with ample opportunities for research and progress in the future. Despite the use of advanced techniques for the detection of human leukocyte antigen (HLA) or non-HLA donor-specific antibodies, alloimmune response remains an important barrier for successful long-term allograft function. Treatment of AMR with currently available therapies has produced a variety of results, some of them suboptimal, precluding the development of standardized protocols. New therapies are promising, but randomized controlled trials are needed to find surrogate markers and improve the efficacy of therapy.

AJKD Atlas of Renal Pathology: Systemic Sclerosis

Systemic sclerosis (scleroderma) has a strong predilection in women, with onset typically between ages 30-50 years. Scleroderma kidney disease occurs in 60%-70% of patients with systemic scleroderma, mostly in those with a diffuse, rather than limited, form of cutaneous scleroderma. Kidney disease is evident by hypertension, proteinuria, and loss of glomerular filtration rate. Scleroderma kidney crisis develops in about 10%-20%, and presents as new onset malignant hypertension with acute kidney injury. Antinuclear antibody is positive in >90% of these patients. Anti-topoisomerase 1 and anti-RNA polymerase antibodies are often associated with kidney involvement. Patients with scleroderma kidney crisis may require dialysis. Use of angiotensin-converting enzyme inhibitors has improved prognosis of patients with kidney crisis, but mortality at 5 years remained at 30%-40%. Light microscopy: Medium- and large-sized arteries are affected by thrombotic microangiopathy. Fibrin thrombi with fibrinoid necrosis of the vessel wall without associated wall inflammation are characteristic. Intimal mucoid change, endothelial swelling, and fragmented red blood cells within the vessel wall occur acutely. Later, there is intimal proliferation, resulting in an “onion skin” concentric appearance with luminal narrowing. Glomeruli typically show ischemic collapse, but may also have mesangiolysis and fibrin thrombi. Glomerular basement membrane duplication is present in chronic cases. Tubular injury occurs with acute injury, and tubular atrophy and interstitial fibrosis are proportional to vascular injury in the chronic stages. Immunofluorescence microscopy: Nonspecific staining for immunoglobulin M and C3 may be seen. Fibrinogen staining can be observed in glomeruli and arteries associated with fibrinoid necrosis or acute thrombotic microangiopathy. Electron microscopy: Endothelial swelling and expansion of lamina rara interna are present. Glomerular basement membrane duplication with cellular interposition is present in chronic stages. Injury from scleroderma is related to endothelial injury of uncertain etiology. About half of patients have anti-endothelial antibodies. Endothelial injury leads to altered vascular permeability and platelet aggregation. Increased renin production exacerbates hypertension. Thrombotic microangiopathy of other causes has similar morphologic findings, including malignant hypertension, drug etiology, complement dysregulation, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura (HUS/TTP), and transplant rejection. Scleroderma mostly affects interlobular arteries in contrast to HUS/TTP, which predominantly affects glomeruli. Malignant hypertension-associated thrombotic microangiopathy may have an identical appearance to that seen in scleroderma, although this is more often associated with underlying arterionephrosclerosis. Clinical history and serologic data help to distinguish these etiologies. •Fibrin thrombi with fibrinoid necrosis, mostly in interlobular arteries•Interlobular arteries with “onion skin” concentric appearance with luminal narrowing in chronic stage•Endothelial injury by electron microscopyFigure 2Scleroderma with artery showing early organization with “onion skin” change caused by lamellation and mucoid change with swelling of the intimal layer. There is corrugation of the glomerular basement membrane (Jones silver stain).Reproduced with permission from AJKD 34(2):e4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Scleroderma with early arterial changes with swollen endothelium and mucoid appearance of the subintima with red blood cell fragments (Jones silver stain).Reproduced with permission from AJKD 34(2):e4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Scleroderma in a chronic stage with fibrous organization of intimal injury resulting in a lamellated “onion skin” appearance of arteries (periodic acid–Schiff stain).Reproduced with permission from AJKD 34(2):e4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Scleroderma with corrugation and expansion of the lamina rara interna, evidence of endothelial injury and ischemia (electron microscopy).Reproduced with permission from AJKD 34(2):e4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Development and validation of a prognostic index for allograft outcome in kidney recipients with transplant glomerulopathy

We studied 92 patients with transplant glomerulopathy to develop a prognostic index based on the risk factors for allograft failure within five years of diagnosis (Development cohort). During 60 months (median) follow up, 64 patients developed allograft failure. A chronic-inflammation score generated by combining Banff ci, ct and ti scores, serum creatinine and proteinuria at biopsy, were independent risk factors for allograft failure. Based on the Cox model, we developed a prognostic index and classified patients into risk groups. Compared to the low risk group (median allograft survival over 60 months from diagnosis), patients in the medium risk group had a hazard ratio of 2.83 (median survival 25 months), while those in the high risk group had a hazard ratio of 5.96 (median survival 3.7 months). We next evaluated the performance of the prognostic index in an independent external cohort of 47 patients with transplant glomerulopathy (Validation cohort). The hazard ratios were 2.18 (median survival 19 months) and 16.27 (median survival 1.6 months), respectively, for patients in the medium and high risk groups, compared to the low risk group (median survival 47 months). Our prognostic index model did well in measures of discrimination and calibration. Thus, risk stratification of transplant glomerulopathy based on our prognostic index may provide informative insight for both the patient and physician regarding prognosis and treatment.

AJKD Atlas of Renal Pathology: Chronic Antibody-Mediated Rejection

Chronic antibody-mediated rejection is commonly associated with progressive loss of kidney function and proteinuria, although early stages may be subclinical and recognizable only through protocol biopsies. Diagnostic criteria according to the Banff 2013 classification (Haas et al, Am J Transplant 2014:14(2):272-283) include the presence of all of the following:1.Histologic evidence of chronic tissue injury, defined by the presence of at least one of the following:a.Transplant glomerulopathy (cg > 0) in the absence of chronic thrombotic microangiopathyb.Severe peritubular capillary basement membrane multilayering by electron microscopyc.New-onset arterial intimal fibrosis with no other known etiology2.Current/recent histologic evidence of antibody interaction with vascular endothelium, defined by the presence of at least one of the following:a.Linear C4d staining in peritubular capillaries (at least 10% by immunofluorescence [IF] on frozen sections or any positivity by immunohistochemistry on paraffin sections)b.At least moderate microvascular inflammation (g + ptc > 2)c.Increased expression of tissue gene transcripts indicative of endothelial injury3.Presence of donor-specific antibodies (DSA; HLA or non-HLA) in the serum Similar to acute antibody-mediated rejection, endothelial injury results from interaction between DSA with primarily mismatched HLA or non-HLA molecules. Repetitive and chronic endothelial damage is associated with new basement membrane formation, which in glomeruli manifests as glomerular basement membrane duplication (transplant glomerulopathy), often with resulting segmental sclerosis, and multilayering of the peritubular capillary basement membrane. Podocyte injury and foot process effacement are common findings. Glomerular basement membrane duplication is also found in other conditions associated with chronic endothelial damage, causing a membranoproliferative pattern of injury. Chronic thrombotic microangiopathy may be indistinguishable from transplant glomerulopathy on a morphologic basis unless features of concomitant acute thrombotic microangiopathy or vascular changes are present. In such cases, correlation with clinical history will be important. Membranoproliferative glomerulonephritis due to immune complex processes (lupus nephritis, immunoglobulin A nephropathy, cryoglobulinemic glomerulonephritis, etc) must be ruled out by IF and electron microscopy. Peritubular capillary basement membrane multilayering, especially if not severe (<7 layers), is not specific to chronic antibody-mediated rejection. Arterial intimal fibrosis is a common finding, often associated with chronic hypertension and aging. Scattered T cells and foam cells may be present in the expanded intima, but are not specific for chronic antibody-mediated rejection and may also be seen in chronic T-cell–mediated rejection. •Transplant glomerulopathy•Peritubular capillary basement membrane multilayering•Peritubular capillary C4d stainingFigure 2Transplant glomerulopathy with prominent glomerular basement membrane duplication and cellular interposition in a capillary loop. Podocytes show significant foot process effacement (electron microscopy).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Chronic antibody-mediated rejection with severe peritubular capillary multilayering (>7 layers of basement membrane), representing response to repetitive and chronic endothelial injury. Note that the endothelial cell is swollen and has lost its normal fenestrations (electron microscopy).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Chronic antibody-mediated rejection with diffuse linear C4d staining in peritubular capillaries (immunofluorescence microscopy, C4d).View Large Image Figure ViewerDownload Hi-res image Download (PPT)

HLA-DR and -DQ Eplet Mismatches and Transplant Glomerulopathy: A Nested Case–Control Study

We conducted a nested case-control study from a cohort of adult kidney transplant recipients to assess the risk of transplant glomerulopathy (TG) as a function of donor and recipient HLA-DR and -DQ incompatibility at the eplet level. Cases (n = 52) were defined as patients diagnosed with transplant glomerulopathy based on biopsies showing glomerular basement membrane duplication without immune complex deposition. Controls (n = 104) with a similar follow-up from transplantation were randomly selected from the remaining cohort. HLAMatchmaker was used to ascertain the number of DRB1/3/4/5, DQA1 and DQB1 related eplet mismatches (eplet load). Multivariable conditional logistic regression models demonstrated an increase in the odds of TG (odds ratios [OR] of 2.84 [95% confidence interval (CI): 1.03, 7.84] and 4.62 [95% CI: 1.51, 14.14]) in the presence of 27-43 and >43 HLA-DR + DQ related eplet mismatches versus <27 eplet mismatches, respectively. When the eplet load was modeled as a continuous variable, the OR for TG was 1.25 (95% CI: 1.04, 1.50) for every 10 additional HLA-DR + DQ eplet mismatches. Our study suggests that minimization of HLA-DR + DQ eplet mismatches may decrease the incidence of transplant glomerulopathy diagnosed by indication biopsies. The role of eplet immunogenicity/antigenicity as determinants of allograft outcomes requires further study.

Class II HLA Eplet Load Is a Risk Factor for Transplant Glomerulopathy.

Study purpose: The objective of this study was to assess the risk of developing transplant glomerulopathy as a function of donor and recipient class II HLA incompatibility at the eplet level. Methods: We conducted a nested case-control study from a cohort of kidney transplant recipients (KTR) transplanted at the University Health Network (UHN) January 1, 2000 to December 31, 2012. Cases (n=52) were defined as adult KTR diagnosed with transplant glomerulopathy based on kidney allograft biopsies showing glomerular basement membrane duplication without immune complex deposition. Controls (n=104) were randomly selected from the remaining cohort at the time of case diagnosis. HLAMatchmaker was used to ascertain the number of class II (DR and DQ) eplet mismatches, or the class II “eplet load,” from donors and recipients' 4-digit HLA typing. DRB1, DR3/4/5, DQA, and DQB typing was updated using molecular methods when absent. Class II eplet load was modeled both as a categorical (DR and DQ eplet load: <27, 27 to 43, >43) and continuous variable. We fit conditional logistic regression models adjusting for recipient, donor, and transplant characteristics to assess the association between transplant glomerulopathy and eplet load. Results: A significant increase in the odds of transplant glomerulopathy was observed across ascending categories of eplet load. The fully adjusted model showed odds ratios of transplant glomerulopathy of 2.84 (95% confidence interval: 1.03, 7.84) and 4.62 (95% CI: 1.51, 14.14) in the presence of 27 to 43 and >43 class II eplet mismatches vs. <27 eplet mismatches, respectively. When the eplet load was modelled as a continuous variable, the odds ratios for transplant glomerulopathy were 1.25 (95% CI: 1.04, 1.50), 1.63 (95% CI: 1.14, 2.33), and 1.27 (95% CI: 0.95, 1.69) for every 10 additional class II (DR and DQ), DR, and DQ eplet mismatches, respectively. Conclusions: Class II eplet load is an independent risk factor for transplant glomerulopathy. Minimization of class II HLA mismatches between donors and recipients at the eplet level and tailoring of immunosuppression regimens to eplet load may decrease the incidence of transplant glomerulopathy in kidney transplant recipients.

A Novel Mouse Model of Podocyte Depletion

Aim: The goal of this study was to examine the capacity for glomerular repair after a podocyte-depleting injury. Methods: We created transgenic (TG) mice expressing the yeast enzyme cytosine deaminase specifically in glomerular podocytes. In these TG animals, the prodrug 5-flucytosine (5-FC) is converted to 5-fluorouracil and promotes cell death. Results: Treatment with increasing dosages of 5-FC caused graded increases in proteinuria 1–2 weeks after treatment, which returned to control levels by the 10-week time point. Light microscopic examination revealed minimal pathology at the 2-week time point, but electron microscopy revealed found foot process effacement as well as focal areas of glomerular basement membrane duplication, and immunohistochemical studies detected podocyte apoptosis and a decrease in the number of Wilms’ tumor protein 1 (WT1)-positive cells. By the 10-week time point, however, the number of WT1-positive cells was similar to controls and a few mice had developed focal areas of glomerulosclerosis. Consistent with the effects of 5-FC on podocyte number, expression of the podocyte mRNAs for nephrin, podocin, synaptopodin and podocalyxin were altered in a similar temporal fashion. Conclusion: The glomerulus has a significant capacity for repair after a podocyte-depleting injury.

Protective effect of lycopene on deltamethrin-induced histological and ultrastructural changes in kidney tissue of rats

Deltamethrin is globally used in crop protection and control of malaria and other vector-borne diseases. It has a potent insecticidal activity with an appreciable safety margin. However, a number of studies have demonstrated nephrotoxicity of deltamethrin in mammalian and nonmammalian species. Lycopene, a carotenoid occurring naturally in tomatoes, has attracted considerable attention as an antioxidant. This study was focused on investigating the possible protective effect of coadministration of lycopene on deltamethrin toxicity. In this study, male albino rats were divided into four groups of 10 animals each: group I served as control, which received standard diet; group II received oral administration of deltamethrin (1.28mg/kg per day) for 30days; group III received both deltamethrin and lycopene (1mg/kg per day); group IV received lycopene (1mg/kg per day). After the experiment, the animals were anesthetized and the cytokine, tumor necrosis factor-α (TNF-α), in the serum was measured; the kidney was taken for histological and ultrastructural studies. Deltamethrin significantly increased the TNF-α. The histopathological examination of kidney showed mild necrotic changes. Ultrastructural changes in renal proximal tubules of deltamethrin-treated group included an increased number and irregular shape of mitochondria with sparse fragmented cristae, serious ultrastructural lesions in renal proximal tubular lining cells, vacuolar degeneration in the epithelial cells, increased number of lysosomes and loss of apical microvilli. In addition, focal segmental thickening and the duplication of glomerular basement membrane and podocyte changes were observed. Histopathological and ultrastructural study showed some protective effect of lycopene on kidney tissues.

Transplantation With Pathologic Kidneys to Improve the Pool of Donors

Kidney transplantation is the best treatment for patients with end-stage renal disease (1), but donors are lacking and waiting lists are increasing in many countries. Living donors, expanded criteria donors, and donations after cardiac death are three ways currently used to increase the pool of donors. Another possibility is to use pathologic organs. Indeed, kidneys with diseases induced by extrarenal mechanisms could recover after transplantation in a new environment. Here, we report an example of this potential strategy. Fourteen months after beginning hemodialysis, a 19-year-old man with Alport syndrome was admitted for a potential transplantation. The donor was a 17-year-old man with a history of a ventriculoperitoneal shunt for hydrocephalus caused by an aqueduct stenosis. He had no underlying renal disease. The cause of death was recurrent hydrocephalus. A kidney biopsy was performed at the time of kidney removal because of a proteinuria (9.2 g/L; estimated glomerular filtration rate [eGFR]=94 mL/min/1.73 m2 using the modification of diet in renal disease formula). Mild and focal glomerular basement membrane duplication was observed in 14 of 17 glomeruli (see Fig. 1). An immunofluorescence study revealed C3 and IgM deposition. This was compatible with an early stage of shunt nephritis (2). At the time of transplantation, no donor-specific antibodies were identified in the recipient using Luminex technology. The flow cytometric crossmatch on T and B lymphocytes for IgG antibodies was negative. We hypothesized that the renal lesions would resolve in a new environment, and transplantation was performed. The recipient received basiliximab and a short course of methylprednisolone for induction and then tacrolimus and mycophenolate mofetil for maintenance therapy. The renal outcome was favorable with immediate output of urine and restoration of graft function. Fifteen days after transplantation, the eGFR was 57 mL/min/1.73 m2 and the proteinuria/creatininuria ratio was 145 mg/mmol. Six weeks after transplantation, the serum creatinine level increased by 20% compared with baseline, and a kidney biopsy was performed (proteinuria=0 g/L). Histopathologic analysis showed mild tubulitis and mild interstitial inflammation (i1, t1, g0, v0, cpt0, ci0, ct0, cg0, mm0, cv0, and ah0), and an immunofluorescence study was negative for C4d, IgM, and C3 deposits, leading to the diagnosis of borderline rejection. None of the nine glomeruli had lesions of membranoproliferative glomerulonephritis. The patient received three pulses of methylprednisolone (500 mg/day). Three months posttransplantation, a protocol kidney biopsy was performed. It contained 14 glomeruli and showed mild interstitial fibrosis and tubular atrophy (i0, t0, g0, v0, cpt0, ci1, ct1, cg0, mm0, cv0, and ah0). An immunofluorescence study was negative for C4d, IgM, and C3 deposits. Six months posttransplantation, the eGFR was 62 mL/min/1.73 m2 and the proteinuria/creatininuria ratio was 17 mg/mmol.FIGURE 1: Histopathologic analysis (PAS). (A) Preimplantation biopsy, showing mild and focal glomerular basement membrane duplication, with normal degree of mesangial cellularity. Taken together with clinical history and IgM and C3 deposition found on immunofluorescence study, these lesions were compatible with an early stage of shunt nephritis. (B, C). Day 40 and day 90 biopsies, showing normal degree of mesangial cellularity (mm0) and no glomerular basement membrane duplication (cg0).This is the first reported case of kidney transplantation from a donor with shunt nephritis. As we had hypothesized, the glomerular lesions were reversible and the graft outcome was good, likely because the kidney was extracted from a deleterious immunologic environment of the donor (3). We cannot rule out the potential role of immunosuppressive treatment on this outcome (4). In conclusion, kidneys with diseases induced by immune-mediated extrarenal mechanisms may be considered for transplantation, after histological evaluation in emergency. This strategy could increase the pool of donors. Florent Guerville Sebastien Lepreux Delphine Morel Thomas Bachelet Karine Moreau Pierre Merville Lionel Couzi Department of Nephrology CHU Bordeaux Hôpital Pellegrin Bordeaux, France Université de Bordeaux CNRS UMR 5164 Bordeaux, France Department of Pathology CHU Bordeaux, Hôpital Pellegrin Bordeaux, France

Overlapping pathways to transplant glomerulopathy: chronic humoral rejection, hepatitis C infection, and thrombotic microangiopathy

Transplant glomerulopathy (TG) has received much attention in recent years as a symptom of chronic humoral rejection; however, many cases lack C4d deposition and/or circulating donor-specific antibodies (DSAs). To determine the contribution of other causes, we studied 209 consecutive renal allograft indication biopsies for chronic allograft dysfunction, of which 25 met the pathological criteria of TG. Three partially overlapping etiologies accounted for 21 (84%) cases: C4d-positive (48%), hepatitis C-positive (36%), and thrombotic microangiopathy (TMA)-positive (32%) TG. The majority of patients with confirmed TMA were also hepatitis C positive, and the majority of hepatitis C-positive patients had TMA. DSAs were significantly associated with C4d-positive but not with hepatitis C-positive TG. The prevalence of hepatitis C was significantly higher in the TG group than in 29 control patients. Within the TG cohort, those who were hepatitis C-positive developed allograft failure significantly earlier than hepatitis C-negative patients. Thus, TG is not a specific diagnosis but a pattern of pathological injury involving three major overlapping pathways. It is important to distinguish these mechanisms, as they may have different prognostic and therapeutic implications.

Early Ultrastructural Changes in Renal Allografts: Correlation With Antibody-Mediated Rejection and Transplant Glomerulopathy

Transplant glomerulopathy (TG) is associated with antibody-mediated renal allograft rejection (AMR) and reduced graft survival. Histologically, TG is typically seen >1 year posttransplantation. However, ultrastructural changes including glomerular endothelial swelling, subendothelial widening and early glomerular basement membrane duplication are associated with development of TG but appear much earlier. We examined the specificity of these changes for AMR, and whether these are inevitably associated with development of TG. Of 98 for cause renal allograft biopsies carried out within 3 months of transplantation with available serologic data, 17 showed C4d-positive AMR and 16 had histologic changes of AMR and donor-specific antibodies (DSA), but no C4d. All three ultrastructural changes were seen in 11 of 17 biopsies with C4d-positive AMR, 8 of 16 with histologic changes of AMR and DSA but no C4d, and 0 of 65 without histologic changes of AMR and/or DSA (p < 0.0001 for both of the former groups vs. the latter). Twenty patients with positive DSA (18 with histologic changes of AMR and 11 C4d-positive) had ≥1 follow-up biopsy; eight developed overt TG 3.5-30 months posttransplantation. Among the 18 patients with DSA and histologic changes of AMR, 11 C4d-positive and 7 C4d-negative, treatment for AMR after the early biopsy significantly reduced subsequent development of overt TG.

Pathogenesis of Transplant Glomerulopathy

Transplant glomerulopathy is a late complication of renal transplantation. The characteristic morphology of transplant glomerulopathy includes thickening of glomerular capillary loops with double contour, and duplication of glomerular basement membrane on electron microscopy. Clinical and experimental evidences support the role of antibody-mediated immune mechanism in the development of transplant glomerulopathy. Antibody-induced endothelial cell injury is the key pathogenesis of transplant glomerulopathy. The evolution of transplant glomerulopathy in the context of immunologic injury is briefly reviewed.

Renal Thrombotic Microangiopathy in Mice with Combined Deletion of Endocytic Recycling Regulators EHD3 and EHD4

Eps15 Homology Domain-containing 3 (EHD3), a member of the EHD protein family that regulates endocytic recycling, is the first protein reported to be specifically expressed in the glomerular endothelium in the kidney; therefore we generated Ehd3 –/– mice and assessed renal development and pathology. Ehd3 –/– animals showed no overt defects, and exhibited no proteinuria or glomerular pathology. However, as the expression of EHD4, a related family member, was elevated in the glomerular endothelium of Ehd3 –/– mice and suggested functional compensation, we generated and analyzed Ehd3 –/–; Ehd4 –/– mice. These mice were smaller, possessed smaller and paler kidneys, were proteinuric and died between 3–24 weeks of age. Detailed analyses of Ehd3 –/–; Ehd4 –/– kidneys demonstrated thrombotic microangiopathy (TMA)-like glomerular lesions including thickening and duplication of glomerular basement membrane, endothelial swelling and loss of fenestrations. Other changes included segmental podocyte foot process effacement, mesangial interposition, and abnormal podocytic and mesangial marker expression. The glomerular lesions observed were strikingly similar to those seen in human pre-eclampsia and mouse models of reduced VEGF expression. As altered glomerular endothelial VEGFR2 expression and localization and increased apoptosis was observed in the absence of EHD3 and EHD4, we propose that EHD-mediated endocytic traffic of key surface receptors such as VEGFR2 is essential for physiological control of glomerular function. Furthermore, Ehd3 –/–; Ehd4 –/– mice provide a unique model to elucidate mechanisms of glomerular endothelial injury which is observed in a wide variety of human renal and extra-renal diseases.

Determinants of Long-Term Graft Outcome in Transplant Glomerulopathy

Transplant glomerulopathy (TG) is a renal allograft disease defined by glomerular basement membrane duplication with peritubular capillary basement membrane multilayering (PTCML), and associated with anti-human leukocyte antigen antibodies and C4d. Outcome in TG is poor but variable, and prognostic factors, particularly those affecting long-term outcome, are not well known. We investigated several potentially prognostic clinical and pathologic factors in TG and evaluated estimated glomerular filtration rate (eGFR) slopes to assess graft function and early decline. We examined all cases of TG from 2001 to 2005 with at least 4-year follow-up after biopsy, excluding those with a second confounding diagnosis. Among 36 cases of pure TG, mean graft age at biopsy was 8.8±6 years. C4d stain was positive in 11 (33%) cases. Clinical characteristics at biopsy were not different based on C4d. C4d was associated with greater PTCML (P=0.03), peritubular capillaritis (P=0.04), and glomerulitis (P=0.03). Death-censored graft survival was significantly associated with interstitial fibrosis (P=0.001), PTCML (P=0.001), and arteriolar hyalinosis (P=0.007), and it showed a trend with proteinuria (P=0.07) and C4d positivity (P=0.08). C4d-positive cases also showed a trend toward rapid graft loss. Analysis of eGFR slopes showed a pattern of preserved, slightly negative slope from transplant until approximately 1 year before biopsy, at which point the slope became significantly more negative (P<0.001). Interstitial fibrosis, PTCML, and arteriolar hyalinosis were significant predictors of graft survival in TG. C4d positivity was associated with a more rapid rate of function decline. eGFR slope data showed significant deterioration in graft function well before the diagnostic biopsy.

The spectrum of FSGS: does pathology matter?

The spectrum of podocytopathies ranges from steroid-responsive minimal change disease (MCD) to devastating focal segmental glomerulosclerosis (FSGS), which may recur and cause end-stage renal disease in the transplant. Sclerotic lesions of glomeruli may also develop as a secondary event, both in immune complex and non-immune diseases, such as conditions with extensive loss of renal mass, diabetic nephropathy, arterionephrosclerosis or reflux nephropathy. Even amongst the group of patients with so-called ‘primary FSGS’, there is increasing recognition of a variety of contributing aetiologies, including genetic, viral, drug toxicity and others [1]. Discernment of clues to patient course and prognosis could be valuable in stratifying patients according to risk and targeting improved treatment. In addition to the traditional methods afforded by renal biopsy, novel approaches with proteomics, microarray gene expression, genetic influences and others have been used to approach this problem [2–4]. Ultimately, of course, patients do not read textbooks, and we must continue to learn from observations in our patients to further refine and improve our approaches for better understanding of these entities. In this regard, the studies of Canaud et al. are of interest [5]. They have studied retrospectively a group of 77 patients with idiopathic nephrotic syndrome and ‘FSGS’ in native kidneys who underwent renal transplantation. Of this largely paediatric cohort, 42 patients had development of nephrotic range proteinuria after transplantation. As expected, early biopsies performed in these patients with recurrent nephrotic syndrome showed no lesions by light microscopy in most patients (32/33). One of these early recurrent nephrotic syndrome biopsies showed perihilar variant FSGS. This constellation of lack of sclerosis in a setting of presumed recurrent disease clinically was a condition unfortunately designated as ‘minimal change disease’ by the authors (see below). Over the long-term follow-up, a total of 20 patients had FSGS lesions in the transplant, and of these, seven showed collapsing lesions at time points ranging from 6 to 48 months after transplant (on average 22 months). Biopsies done at month 3 in 40 of the patients showed lesions in 14 patients. Of interest, lesions observed in month 3 biopsies were the same as seen later in seven of the eight patients who had segmental lesions at both time points, with one patient showing transition from a tip lesion to a perihilar lesion. Patients who showed the same segmental lesions present at both time points included collapsing (n = 2), cellular (n = 3) and perihilar (n = 2) variants. It is important in analysing segmental lesions in the transplant to recognize that the transplanted kidney remains, of course, a kidney, and is subject to all injuries that may affect the native kidney. In this report, unfortunately, electron microscopy was not done to assess these lesions. This is important in light of the multiplicity of injury that may result in segmental lesions. Recent studies have demonstrated that secondary segmental sclerotic lesions have subtotal foot process effacement, often <50%, whereas primary FSGS typically has extensive foot process effacement [6,7]. Thus, to completely understand the dynamics of the collapsing and perihilar lesions observed, particularly at late time points after transplant, it would be of great interest to examine the extent of foot process effacement and other concomitant lesions that could suggest e.g. calcineurin inhibitor toxicity as a contributor to the collapsing phenotype. Our recent survey of transplant lesions demonstrated that recurrence of the disease FSGS in the transplant (diagnosed based on significant proteinuria and extensive foot process effacement, with or without overt segmental sclerosis) was very rare after 2 years, and most recurrent FSGS occurred within the first 6 months after transplantation [8]. Thus, segmental sclerotic lesions at later time points most often represented transplant glomerulopathy evidenced by duplication of glomerular basement membrane and increased lamina rara interna and subtotal foot process effacement by electron microscopy; or calcineurin inhibitor toxicity, often with collapsing features and subtotal foot process effacement, with concomitant arteriolar nodular hyalinosis [9]. New approaches to investigate the aetiology of this spectrum of MCD to FSGS lesions include immunohistochemistry, proteomic and gene array studies. Thus, the group of Kerjaschki has demonstrated that dystroglycan staining is markedly decreased in minimal change disease, while remaining intact in the non-sclerotic areas in FSGS [4]. Proteomic and gene array studies by Kretzler also point to a different pathogenesis of minimal change disease and FSGS [2]. The term ‘minimal change disease’ is typically used for a steroid-responsive

Ultrastructural Examination of Glomerular and Tubular Changes in Renal Allografts with Cyclosporine Toxicity

The introduction of cyclosporine (CsA) has improved the clinical results of renal transplantation significantly; however, these improvements were closely associated with an increased incidence of renal dysfunction. The present study sought to examine the ultrastructural changes in renal allografts with CsA nephrotoxicity. Nine patients who underwent renal transplantation at the Baskent University Faculty of Medicine between 2001 and 2002 were examined; 26 biopsies of these nine patients who had received their first renal allograft were included in this study. All patients with CsA toxicity showed some form of glomerular endothelial cell injury. The swelling of mitochondria was present in three of nine renal allografts with CsA toxicity, and podocyte changes were found significantly more frequently among patients with CsA toxicity. In addition, focal segmental thickening and the duplication of glomerular basement membrane were observed statistically more frequently. In conclusion, these findings could help differentiate CsA toxicity from other conditions and develop better treatment strategies.