Electron-dense Deposits Research Articles

P0477CLINICOPATHOLOGICAL STUDY OF MIXED CRYOGLOBULINEMIC GLOMERULONEPHRITIS SECONDARY TO HEPATITIS B VIRUS INFECTION

Abstract Background and Aims To describe the clinical features, renal pathology findings and prognosis in patients with mixed cryoglobulinemic glomerulonephritis (Gn) caused by hepatitis B virus (HBV) infection. Method This was a retrospective study including seven Chinese patients with HBV infection associated mixed cryoglobulinemic Gn in a tertiary referral hospital from April, 2016 to March, 2019. The demographic, clinical, pathological characteristics, treatment and follow-up data were collected and analyzed. Results Age at renal biopsy was 47±12 years, including three females and four males. 24hUP was 5.6(3.0, 6.6)g/d and five cases presented with nephrotic syndrome. The median baseline eGFR(CKD-EPI) was 23.5(20.2, 46.3) ml/min per 1.73m2. The extrarenal manifestations were: purpura (n=6), arthralgia (n=1), peripheral neuropathy (n=1), and cardiomyopathy (n=1). Six cases had type II cryoglobulinemia with IgMκ, the other one had type III. The median cryocrit was 4.0 (1.0, 15.0) %, rheumatoid factor was 368(117, 733) IU/ml, C3 was 0.48(0.41, 0.57) g/L, C4 was 0.013(0.003, 0.118) g/L. Renal pathologic findings on light microscopy: endocapillary proliferative Gn (n=3), membranoproliferative Gn (n=3), and mesangial proliferative glomerulonephritis(n=1). Hyaline thrombi were seen in four cases, while crescents were found in two cases. On immunofluorescence microscopy, the predominant types of immunoglobulin deposits were: IgM(n=5), IgA(n=1), and codominance of IgG and IgA(n=1). HBsAg and HBcAg deposits were found in only one case. Ultrastructural studies showed granular subendothelial and mesangial electron-dense deposits in all patients and organized microtubules was seen in only one case. All patients received antiviral medication (entecavir, n=6; lamivudine, n=1). They were given corticosteroid alone(n=2) or combined with cyclophosphamide(n=4) or mycophenolate mofetil(n=1). Two patients received plasmapheresis. The median follow-up time were 18 (6, 37) months. One patient died from pneumonia, and one progressed to end stage of renal disease (ESRD). At endpoint of follow-up, 24hUP was 2.1 (0.8-5.2) g/d, and eGFR (CKD-EPI) was 55.3 (20.7, 111.8) ml/min per 1.73m2. Conclusion Mixed cryoglobulinemic Gn should be screened for HBV etiologies, especially in HBV-endemic country. Endocapillary proliferative Gn was the common pathologic type, as well as membranoproliferative Gn. Diagnosis and treatment in early stage benefit patients’ renal outcome. Long-term prognosis should be investigated in further studies.

Treatment-resistant nephrotic syndrome in dense deposit disease: complement-mediated glomerular capillary wall injury?

BackgroundThe C3 glomerulopathies (C3G) are recently defined glomerular diseases, attributed to abnormal complement regulation. Dense deposit disease (DDD) is part of the spectrum of C3G, characterized by electron-dense deposits in the lamina densa of the glomerular basement membrane. Patients with DDD present with hematuria, variable degrees of proteinuria, and kidney dysfunction. Kidney biopsies typically disclose proliferative and inflammatory patterns of injury. Treatment with glucocorticoids and mycophenolate mofetil has been shown to achieve remission of proteinuria in a significant proportion of C3G patients.Case-diagnosis/treatmentWe report two patients with persistent nephrotic syndrome while on immunosuppressive therapy. Repeat kidney biopsies disclosed massive C3 deposits with foot process effacement in the absence of proliferative or inflammatory lesions on light microscopy.ConclusionThese cases, coupled with data from animal models of disease and the variable response to eculizumab in C3G patients, illustrate that two different pathways might be involved in the development of kidney injury in C3G: a C5-independent pathway leading to glomerular capillary wall injury and the development of proteinuria versus a C5-dependent pathway that causes proliferative glomerulonephritis and kidney dysfunction.

Renal hypoplasia can be the cause of membranous nephropathy-like lesions.

Renal hypoplasia (RH) is the most common cause of chronic kidney disease in children. In cases of RH, proteinuria is often induced by glomerular hypertrophy and hyperfiltration that is commonly associated with focal segmental glomerulosclerosis. This study reports the first case series of a possible association between RH and membranous nephropathy (MN). Of the 168 children with RH who visited our department between 1999 and 2017, five with overt proteinuria (≥ 1g/gCr) underwent renal biopsy. We retrospectively reviewed the medical charts and analyzed biopsy specimens using light microscopy (LM), immunofluorescence (IF), and electron microscopy. The five children (four boys and one girl) had a median age of 5.5years at the time of renal biopsy. The median proteinuria was 4.23g/gCr (range 1.46-14.25), median serum albumin, 2.9g/dL (range 2.3-3.7), and median estimated glomerular filtration rate, 59.7mL/min/1.73m2 (range 36.7-103.6). LM showed segmental spike formation and mesangial hypercellularity and IF study showed segmental granular immunoglobulin G (IgG) staining (IgG1 and IgG3 dominant) along the capillary loops in all five patients. Electron-dense deposits were observed in the subepithelial and mesangial areas. Thus, the pathological studies showed MN-like lesions in all patients. Our study suggests that RH can be the cause of MN-like lesions.

Immunization with Pentraxin3 prevents transition from subclinical to clinical lupus nephritis in lupus-prone mice: Insights from renal ultrastructural findings

BackgroundPentraxin3 (PTX3) is an emerging player in lupus nephritis (LN). Anti-PTX3 antibodies showed to delay LN occurrence in vivo. AimTo evaluate renal changes following immunization with PTX3 in a murine model of LN. Materials and methodsTwenty-two lupus-prone New Zealand Black/White (NZB/W)F1 mice were divided into two groups (n = 11) and subcutaneously injected with human recombinant (hr)PTX3 100 μg or phosphate buffer saline (PBS) 200 μl, three times 3 weeks apart, starting before development of proteinuria. Five mice from each group were scheduled for sacrifice at week 22 and 6 from each group at week 29. Renal lesions included electron-dense deposits (EDD), glomerular deposition of IgG, complement and PTX3 as markers of renal inflammation. They were evaluated by immunofluorescence (IF), confocal and immunoelectron microscopy (IEM). Validated semiquantitative scores were used when available to score renal lesions. Chi-squared test with Fisher exact test was used for comparison. ResultsNineteen out of 22 mice were sacrificed as scheduled. Only hrPTX3-immunized mice developed anti-PTX3 antibodies. Compared to PBS-injected mice, they displayed a dramatic decrease in glomerular deposits of IgG, C1q and PTX3, as well as in the amount of EDD (p = 0.006) and podocyte effacement (p = 0.043). Importantly, PTX3 was pinpointed inside the EDD and co-localized with nuclear material. ConclusionsImmunization with PTX3 prevented progression from the preclinical to the clinical stage of LN, inciting anti-PTX3 antibodies and preventing renal PTX3 deposition. PTX3 is a novel component of EDD, submitting it as one initiating autoantigen in LN and as potential target for early treatment.

SAT-415 SEVERE GLOMERULAR ENDOTHELIAL INJURY AND MILD TO MODERATE PROTEINURIA DUE TO TAFRO SYNDROME WITH RENAL DYSFUNCTION

TAFRO syndrome has been recognized as a severe subtype of idiopathic multicentric Castleman’ s disease(iMCD), and renal dysfunction is a life threatening complication of this disease. We previously reported two case-reports about renal biopsies of TAFRO syndrome, showing endocapillary proliferative glomerulonephritis, but precise mechanism and clinical course have not been elucidated yet. To further evaluate the characteristics of renal dysfunction due to TAFRO syndrome, we conducted a cross-sectional study. The object of patients were either diagnosed as iMCD or TAFRO syndrome who were extracted from 4,248 renal biopsies performed between 2001 and 2018. The renal biopsies were evaluated by light microscopy, immunofluorescence staining, and electron microscopy. Clinical and laboratory data were collected from electrical medical chart. Statistical analysis was conducted by using R version 3.6.1. This observational study was approved by IRB and informed consent was obtained from all living patients. Fifteen cases were eligible for the study, and seven were diagnosed as TAFRO syndrome while eight were as iMCD without TAFRO syndrome (iMCD-NOS). Patients with TAFRO were lower platelet count (36 [28-115]*103/μL in TAFRO syndrome vs 308 [130-424*103/μL] in iMCD-NOS, p-value <0.01), higher serum ALP (641 [534-1438] IU/L vs199 [85-352]IU/L, p<0.01), total protein(5.2 [5.0-6.8] g/dL vs 10.5 [7.7-14.8]g/dL, p<0.01), and lower IgG level(835 [684-1449]mg/dL vs 5249 [2800-8198]mg/dL). Proteinuria in TAFRO syndrome was mild to moderate level and lower than iMCD-NOS, but this was not statistically significant (0.45[0.28-3.71]g/gCre vs 1.07[0.04-5.34]g/gCre, p=0.43). There was no statistical difference in serum creatinine level, serum albumin level and hematuria. In renal biopsy, severe endothelial injury such as endothelial swelling and narrowing of glomerular tuft was commonly observed in TAFRO syndrome (figure), while varieties of histological diagnosis were found in iMCD-NOS; AA amyloidosis (2 cases), membranous nephritis(2 cases), minor glomerular abnormality (2 cases), nephrosclerosis(1 case), IgA deposition(1case), renal sclerosis (1 case). In addition, any deposition was not detected by electron dense deposit or immunofluorescence staining in TAFRO syndrome. Hemodialysis was required for 4 out of 7 patients with TAFRO syndrome and all of them recovered after the initiation of anti IL-6 receptor antibody or anti CD20 monoclonal antibody. On the other hand, 3 out of 8 patients with iMCD-NOS required maintenance hemodialysis despite of anti IL-6 receptor antibody based immunosuppressive therapy.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mild to moderate proteinuria, severe endothelial injury without any deposit were common characteristic of TAFRO syndrome compared with iMCD-NOS. Cytokine targeted immunosuppressive therapy was effective in severe renal dysfunction due to TAFRO syndrome even if hemodialysis was required. The difference in treatment response between TAFRO syndrome and iMCD-NOS might be related to the pathogenesis of glomerular changes of both diseases.

SUN-422 MONOCLONAL IGA NEPHROPATHY WITH LATTICE LIKE PARA-CRYSTALLINE DEPOSITS IN AN ELECTRON MICROSCOPY: A RARE FINDING

Abnormal plasma cells frequently cause the secretion of monoclonal immunoglobulins or immunoglobulin fragments into the serum which leads to various diseases including renal diseases. When patients do not meet the criteria for overt multiple myeloma/B-cell proliferation, and the hematologic disorder is generally consistent with monoclonal gammopathy of undetermined significance (MGUS) but renal abnormalities are consistent, it is called monoclonal gammopathy of renal significance (MGRS). It is associated with high morbidity so early recognition is important, as chemotherapy invariably improves outcomes. Proliferative glomerulonephritis secondary to monoclonal IgA has been previously described on single occasional only and electron microscopy finding is quite unique, with mesangial and subendothelial deposition of lattice-like para crystalline arrays. This is a case report. Case details: A 46-year-old male patient with proteinuria and hematuria for 20 months presented to the nephrology outpatient department. His protein was 3+ and hemoglobin was 2+ in urine. All of the serologies were negative. Renal biopsy was done, which showed mild increase in mesangial matrix with slight accentuation of lobulation, double contours on silver and PAS stains. There were no glomeruli in the original frozen material for immunofluorescence studies; therefore, immunofluorescence was carried out on paraffin sections which showed moderate mesangial IgA staining and mild mesangial lambda staining. Rest of the stains were negative in glomeruli. IgG, IgA, IgM, C3, C1q, kappa, lambda and albumin were used as a panel for immunofluorescence. Electron microscopy showed numerous electron-dense para crystalline deposits in mesangial and subendothelial locations with highly distinctive organized substructure forming lattice-like para crystalline arrays with a periodicity of 18 nm which were slightly curved in some areas. Proliferative glomerulonephritis with monoclonal gammopathy has been reported few times but most of these deposits were granular and rarely fibrillary deposits were present. Among of the cases of renal disease due to monoclonal gammopathy, IgG was the most common immunoglobulin that was deposited, followed by IgM. Renal disorder secondary to a monoclonal IgA lambda is very rare. Deposition of lattice like paracrystalline substructure with parallel arrays in a “blocky” pattern is a unique finding in proliferative glomerulonephritis due to monoclonal lambda restricted IgA nephropathy, which was only reported by Soares et al before this case. Such electron microscopy picture is very rare and unique, and diagnostic for the disease.

SUN-389 Collagenofibrotic glomerulopathy in kidney transplant recipient: A first report

Collagenofibrotic glomerulopathy (CG) also known as collagen type 3 glomerulopathy is a rare disease characterized by the deposition of collagen type 3 in the glomeruli. The absence of skeletal abnormalities in these patient is one feature that helps in distinguishing CG from nail patella syndrome, another collagen type 3 deposition disease in renal glomeruli. High serum levels of pro-collagen type III peptide were reported in this disease. Patients may have proteinuria, hematuria and/or renal dysfunction. CG is considered a progressive disease with variable rate of progression. CG manifests as membranoproliferative pattern of injury by light microscopy with negative immunofluorescence. The definitive diagnosis is made by electron microscopy with the characteristic subendothelial and mesangial curved and comma-like banded collagen type 3 fibers of 45-65 nm in periodicity. A retrospective chart review of collagenofibrotic glomerulopathy case at the American University of Beirut Medical Center. A 39 years-old man had a living-related kidney transplant 13 years ago for unknown cause ESKD. An allograft biopsy was performed for rise in creatinine and proteinuria without hematuria. He has been maintained on cyclosporine, azathioprine and prednisone for 12 years before a switch to tacrolimus, mycophenolate mofetil (MMF) and prednisone one year, done prior for a rise in creatinine at an outside hospital. He was diagnosed with hypertension and diabetes mellitus three years prior to biopsy. His family history is negative for renal disease.Table 1Laboratory values at the time of kidney biopsyCreatinine1.8-2.2 mg/dlTacrolimus4.624 hours urine protein1.2 grams24 hours urine albumin0.65 gramsHbA1c7.8 %HIV serologyNegativeHepatitis B and C serologyNegativeUrinalysis+ 2 proteinuria, negative hematuria Open table in a new tab Table 2Pathologic FindingsLight microscopy (LM), figure 1 (H&E x400).Mesangial matrix expansion and hypercellularity, endocapillary hypercellularity, thickened glomerular capillary wall and focal double contour formation.Immunofluorescence (IF)Negative for IgA, IgG, IgM, C3, C4, C1q, kappa and lambda.Electron microscopy (EM) figure 2Accumulation of collagen fibers between the capillary basement membranes and swollen endothelial cells, with increased mesangial matrix deposition. Tubules show tubular injury with cytoplasmic vacuolization. No subepithelial, subendothelial or mesangial electron dense deposits are identified. The capillary loops are patent. Open table in a new tab Figure 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) We are reporting the first case of post-transplant Collagenofibrotic glomerulopathy (CG). The recurrence post-transplant points towards a systemic disease process, including genetic predilection. It highlights the importance of considering unrecognized kidney disease causes in patients with unknown cause ESKD.

Physiological and ultrastructural responses of the brown seaweed Undaria pinnatifida to triphenyltin chloride (TPTCL) stress

Triphenyltin chloride (TPTCL) is a well-known marine pollutant that may constitute major environmental threats to seaweed mariculture. In the present study, the toxic effects of TPTCL on physiology and ultrastructure of cultivated sporophytes of Undaria pinnatifida were investigated under different TPTCL concentrations ranging from 0 to 100 μg L−1. Significant negative effects of increased TPTCL concentration were detected in the relative growth rates, survival percentages and chlorophyll a contents of young and adult sporophytes. Low TPTCL concentrations could significantly stimulate the activities of enzymes related to nitrogen metabolism. The chloroplast, mitochondria and nucleus inside cells were greatly damaged by TPTCL. Meanwhile, significant increases of electron dense deposits and physodes were found. Additionally, young sporophytes exhibited greater tolerance to TPTCL stress than adult sporophytes. The results of this study indicate that coastal TPTCL pollution could reduce the productivity and quality of cultivated U. pinnatifida.

Demonstration and Characterization of Cyst-Like Structures in the Life Cycle of Trichomonas vaginalis.

Trichomonas vaginalis is the parasitic protozoan residing in human urogenital tract causing trichomoniasis, which is the leading non-viral sexually transmitted disease. It has cosmopolitan distribution throughout the globe and affects both men and women. Lifecycle of the parasite has been traditionally described as consisting of motile and symptom-causing trophozoites. Chemical and temperature perturbations in trophozoites have been shown to aid conversion to pseudocysts, which is poorly investigated. In the current study, we show the formation of viable cyst-like structures (CLS) in stationary phase of T. vaginalis axenic culture. We used a fluorescent stain called calcofluor white, which specifically binds to chitin and cellulose-containing structures, to score for T. vaginalis CLS. Using flow cytometry, we demonstrated and quantitated the processes of encystation as well as excystation; thus, completing the parasite's lifecycle in vitro without any chemical/temperature alterations. Like cysts from other protozoan parasites such as Entamoeba histolytica and Giardia lamblia, T. vaginalis CLS appeared spherical, immotile, and resistant to osmotic lysis and detergent treatments. Ultrastructure of CLS demonstrated by Transmission Electron Microscopy showed a thick electron-dense deposition along its outer membrane. To probe the physiological role of CLS, we exposed parasites to vaginal pH and observed that trophozoites took this as a cue to convert to CLS. Further, upon co- culturing with cells of cervical origin, CLS rapidly excysted to form trophozoites which abrogated the cervical cell monolayer in a dose-dependent manner. To further corroborate the presence of two distinct forms in T. vaginalis, we performed two-dimensional gel electrophoresis and global, untargeted mass spectrometry to highlight differences in the proteome with trophozoites. Interestingly, CLS remained viable in chlorinated swimming pool water implicating the possibility of its role as environmentally resistant structures involved in non-sexual mode of parasite transmission. Finally, we showed that symptomatic human patient vaginal swabs had both T. vaginalis trophozoites and CLS; thus, highlighting its importance in clinical infections. Overall, our study highlights the plasticity of the pathogen and its rapid adaption when subjected to stressful environmental cues and suggests an important role of CLS in the parasite's life cycle, pathogenesis and transmission.

Epitope Mapping of Human α3(IV)NC1-Induced Membranous Nephropathy in Mice

Background and Aim: Primary membranous nephropathy (pMN) is the most common cause of nephrotic syndrome in adults. Recent studies suggested that immunization of DBA/1 mice with the main antigen of antiglomerular basement membrane disease (GBM) disease, α3(IV)NC1, could lead to MN lesions. This study aimed to explore the pathogenic epitopes for mouse MN. Methods: Twenty-four linear peptides were synthesized spanning human α3(IV)NC1. Male DBA/1 mice aged 6–8 weeks were immunized with the peptides 200 μg/mouse in Freund’s complete adjuvant subcutaneously and boosted 3 times with the peptides in Freund’s incomplete adjuvant in weeks 3, 5, and 7. The blood and 24-h urine samples were assessed every 2 weeks. The kidneys were examined when the mice were sacrificed at 18 weeks. Results: All the mice immunized with human α3(IV)NC1 and the 24 peptides produced circulating antibodies against the immunogens at 2 weeks and achieved the maximum titers at 8 weeks. About 5/6 (83%) mice immunized with α3(IV)NC1 and (3/6) 50% of the mice immunized with peptide 23 (α3_141–154) showed proteinuria at 8–10 weeks and increased continuously. The kidneys showed granular depositions of IgG, C3, and C5b-9 along the glomerular capillary loops. The major IgG subclass was IgG1 (equivalent to human IgG4). GBM thickening with the formation of spikes and subepithelial electron-dense deposits were observed under electron microscope. Conclusion: The linear peptide of α3_141–154 could induce clinical and histopathological features of MN in DBA/1 mice, which might give clues to the mechanism of MN in combination with anti-GBM disease.

Monoclonal immunoglobulin mediates complement activation in monoclonal gammopathy associated-C3 glomerulonephritis

BackgroundC3 glomerulonephritis (C3GN) is a rare disease caused by inherited or acquired complement alternative pathway (CAP) dysregulation, which could also be secondary to monoclonal gammopathy of undetermined significance (MGUS). Herein, we described a patient presenting with C3GN and monoclonal gammopathy, and the pathogenic association between the two diseases was further explored in vitro.Case presentationA 76-year-old Chinese man presented with low serum C3 level, haematuria and nephrotic syndrome, and experienced rapid worsening of renal function over a period of 10 months. His serum and urine immunofixation electrophoresis both revealed a monoclonal IgGλ. A bone marrow puncture showed plasma cell dyscrasias with the highest plasma cell count of 5.25%. Kidney biopsy showed the presence of C3 glomerulonephritis, with exclusive deposits of C3 visible on immunofluorescence, a membranoproliferative pattern on light microscopy and electron dense deposits in sub-epithelial, intramembranous, sub-endothelial and mesangial regions by electron microscopy. The patient was positive for C3 nephritic factor (C3NeF) activity and anti-CFH autoantibodies, and all became negative during disease remission. The anti-CFH autoantibodies purified from the patient’s plasma exchange fluids were proven to be a monoclonal IgGλ, and could inhibit CFH binding to C3b and accelerate the formation of C3 convertase indirectly by interfering with the formation-impeding activity of CFH. No deficiency of candidate genes, especially variants in CFH, was detected in our patient. Based on the pathological and laboratory findings, the diagnosis of monoclonal gammopathy of renal significance (MGRS)-associated C3GN was finally made.ConclusionsThis is the first demonstration that intact monoclonal immunoglobulin (IgGλ) could act as an anti-CFH antibody and lead to MGRS-associated C3GN by activating the CAP.

Membranous nephropathy with solitary polyclonal IgA deposition: A case report andliterature review.

A 60-year-old man presented with nephrotic syndrome (NS). Light microscopy of renal biopsy specimens showed minor glomerular abnormalities, while immunofluorescence microscopy revealed solitary polyclonal granular IgA deposition along the glomerular capillary walls. Electron microscopy showed small amounts of electron-dense deposits in the subepithelial area, but not in the mesangial area. In this patient, apparent underlying disease was not found during the 3-year follow-up, and low-dose prednisolone was effective in the treatment of NS. To our knowledge, there is only one case report of membranous nephropathy with clinicopathological features similar to our case.

The importance of tubuloreticular inclusions in lupus nephritis

Tubuloreticular inclusions (TRI) are distinctive cytoplasmic structures of unknown origin that typically associate with autoimmune and viral diseases. We investigated the clinical and prognostic relevance of TRI detection in patients with lupus nephritis (LN). We conducted a single centre study of patients (n=84) with biopsy evidence of LN. Clinical variables included demographics, SLEDAI score, and autoantibody profiling; while histological evaluation included TRI presence, International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification with NIH activity and chronicity indices, immunofluorescence, and other EM findings. Patients with and without TRI were compared by non-parametric statistical methods and survival analysis for the endpoints of death and renal failure. TRI were detected in 37 patients (44%) that were younger (28.4 vs 34.3 years, p=0.02) and more often from Asian background (37.8% vs 19.1%, p=0.04) compared to patients without TRI. SLEDAI score (11 vs 12 units, p=0.36) and amount of proteinuria (370 vs 340 mg/mmol, p=0.71) were similar in both groups; however, TRI positive patients had increased frequency of anti-SSB antibodies (16% vs 2%, p=0.02), 'full house' immune complex deposition (85% vs 58%, p=0.04) and subendothelial electron dense deposits (83% vs 65%, p=0.07), but were less often anti-dsDNA Ab positive (62% vs 85%, p=0.02). Patient and renal survival were not influenced by TRI status. TRI were observed in nearly half of all LN patients and TRI positive patients more often carried anti-SSB antibodies. However, TRI had little bearing on disease presentation or outcome in LN.

Case report: a peculiar glomerulopathy in a patient suffering from nephrotic syndrome

BackgroundPodocyte infolding glomerulopathy (PIG) is a rare histopathologic finding with global infolding of the podocytes into the glomerular basement membrane (GBM), accompanied by microstructures underneath. Described in 2002 for the first time, PIG was proposed as a new pathological entity in 2008 based on the largest case series so far. Yet all of the described cases derive from Asian countries. We report a case from Germany fulfilling the diagnostic criteria of PIG. Considering the scarcity of data on this entity especially in Western countries, collecting cases like ours and multicentric meta-analyses will be crucial to obtain a better understanding of PIG, its causes, clinical course and potential treatment options.Case presentationA 56-year-old Caucasian woman with a history of rheumatoid arthritis (RA), no other comorbidities and no known renal disease was admitted to the hospital with acute kidney injury (AKI) and nephrotic syndrome. Physical examination was unremarkable except for anasarca. Renal ultrasound revealed no abnormalities. Laboratory and urine analyses were consistent with the nephrotic syndrome and renal failure. Serological studies regarding ANA, ANCA, anti-PLA2R autoantibodies, complement, virus infections, immunofixation and quantitative light chain analysis were unremarkable. A renal biopsy was performed. Light microscopic examination showed flattened tubular epithelium consistent with acute tubular damage, no infiltrates and unremarkable glomeruli except diffuse and global holes in the GBM (Fig. 1a) and negative staining for immunoglobulin heavy-chains, light-chains and complement split products. Electron microscopy revealed a rare correlate for these holes: global peculiar infolding of podocyte cytoplasm into the GBM. Most of these infoldings were accompanied by condensation of the GBM underneath. No such condensation or electron dense deposits were found without these infoldings or outside the GBM.ConclusionHere we report the first case of PIG outside of Asia. Since there are only few reports about this specific finding, we feel there is a need to share information in an attempt to accumulate knowledge about this possible new entity and potential treatment options.

Membranoproliferative glomerulonephritis with deposition of monoclonal IgG evolved from polyclonal IgG: a case report with two consecutive renal biopsies

BackgroundProliferative glomerulonephritis with monoclonal Immunoglobulin (G) deposits (PGNMID) is a rare kind of MGRS with intact monoclonal IgG deposition. Seventy percent of PGNMID patients were negative for M-spike.Case presentationA 51-year-old Chinese woman presented with 16-month history of chronic nephritic syndrome. Her first biopsy showed a MPGN pattern, and the IF showed polyclonal IgG deposition but with IgG3λ dominance, MGRS was highly suspected. But the serum/urine IFE and bone marrow examination was negative for monoclonal gammopathy. She was treated with RAS inhibitors, and monitored carefully in the outpatient clinic. When the proteinuria was not controlled by RAS inhibitors, immunosuppressive agents were initiated. The second biopsy was done due to her acute kidney injury 9 months later, showing a MPGN pattern with acute tubulointerstitial disease, but the IF showed monoclonal IgG3λ deposition. The κ light chain, IgG1, IgG2 and IgG4 were absent. Electron microscopic examination revealed electron-dense deposits in the mesangial, subendothelial and subepithelial area which is the same as the first renal biopsy. The final diagnose of this patient was PGNMID (IgG3λ) with non-organized deposits. Repeated serum/urine IFE and free light chain still failed to identify monoclonal gammopathy. The patient was treated with steroid and cyclophosphamide, and her serum creatinine decreased.ConclusionsSome of the PGNMID patients may be derived from polyclonal immune complex mediated glomerulonephritis.

SUN-017 LOW-VACUUM SCANNING ELECTRON MICROSCOPY ANALYSIS FOR HUMAN BIOPSY SAMPLES

One of the major goal of biological microscopy is to elucidate the structural evidence relating functional activity or pathophysiological condition. High resolution imaging by electron microscopy can provide detailed tissue architectures, but the specific and skillful technics are required and it takes long time to the sample preparation. Low-vacuum scanning electron microscopy (LV-SEM) developed recently is a simple and fast technic to observe higher resolution of histological paraffin sections prepared for conventional light microscopy. Periodic acid methenamine silver (PAM) stain is a routine staining for basement membrane in renal biopsy, and contains silver, a heavy metal, which enhances the backscattered electron signal. To evaluate the benefit of LV-SEM, we observed PAM stained glomerular basement membrane (GBM) in human renal biopsy samples. The study was performed under the declaration of Helsinki principles. The study protocol was approved by the Institutional Review Board of the Tohoku Medical and Pharmaceutical University. All participants provided written informed consent after a full explanation of the purpose of the study and the potential risk involved. The human kidneys were obtained from patients with membranous nephropathy, IgA nephropathy, Alport syndrome, or thin basement membrane disease by renal biopsy and embedded in paraffin. Four-μm sections were deparaffinized with xylene, hydrated with ethanol, and stained with periodic acid methenamine silver (PAM). Miniscope TM4000 (Hitachi High-Technologies) was used to observe the GBM by detecting backscattered electron signal. After evacuation of the specimen chamber a few minutes, the sections were observed under the electron beam accelerating voltage of 10 kV for higher magnification (x2,500 to x20,000) or 15 kV for lower magnification (x40 to x2,000) with 30 Pa. The observations at higher magnification were easily performed under LV-SEM by zooming up from x40 to x20,000. The PAM-positive GBMs were bright under LV-SEM. The GBMs showed dark electron dense deposits in membranous nephropathy and IgA nephropathy, and characteristic coarse meshwork appearances in Alport syndrome. The GBMs in thin basement membrane disease seemed relatively thinner than the others. Direct observation of conventional renal biopsy paraffin sections by LV-SEM is a simple and beneficial method for the rapid and detailed analysis of the diseases. It will likely be useful for the observation of GBM morphological change.

Eculizumab deposits in vessel walls in thrombotic microangiopathy

Terminal complement inhibition therapy with eculizumab (a humanized monoclonal antibody to C5) has revolutionized the treatment of patients with thrombotic microangiopathy (TMA). Successful responders are often placed on long-standing therapy to prevent disease recurrence in the native kidney or allograft. The tissue deposition of eculizumab in patients with C3 glomerulopathy has been described but no studies have yet investigated tissue deposition of eculizumab in cases where it was indicated for thrombotic microangiopathy which, unlike C3 glomerulopathy, does not usually show immune-type electron dense deposits. To evaluate this, we reviewed biopsies from 13 patients who received eculizumab for TMA treatment or prevention of recurrence. We found IgG2, IgG4, and kappa positivity within arterioles corresponding to eculizumab deposits, with similar distribution to C5b-9, in all but one patient. In that patient eculizumab therapy had been discontinued 24 months prior to biopsy. Deposits in arterioles could be seen as early as one day after infusion and after a single dose of eculizumab, and were detected up to 162 days after therapy discontinuation. This may play a role in controlling local complement activation-associated vascular changes in these patients. Thus, IgG subclass staining by immunofluorescence is important to avoid misdiagnoses of immune-complex or monoclonal immunoglobulin deposition disease in patients with TMA who received eculizumab.

An IgA1-lambda-type monoclonal immunoglobulin deposition disease associated with membranous features in a patient with IgG4-related kidney disease: a case report

BackgroundIgG4-related disease (IgG4-RD) is a newly recognized fibroinflammatory condition. The kidney is one of the organs commonly affected by IgG4-RD. Tubulointerstitial nephritis (TIN) is the main feature, and membranous nephropathy (MN) has also been described frequently. In MN, polyclonal immunoglobulins and complements are deposited in granular form along the glomerular basement membranes (GBMs). Unusual cases of monoclonal immunoglobulin deposition disease (MIDD) associated with membranous features have been reported. MIDD is morphologically similar to MN but contains immunoglobulins considered to be derived from single B-cell clone.Case presentationWe describe a 65-year-old man who was referred to our hospital because of hyperproteinaemia, eosinophilia, anaemia, and proteinuria. A renal biopsy demonstrated infiltration of plasma cells and eosinophils in the interstitium, and the ratio of IgG4-positive plasma cells to IgG-positive plasma cells was 55%. The patient was diagnosed as having IgG4-related TIN. Periodic acid methenamine silver staining under light microscopy revealed a bubbling appearance and spike formation in the GBM. On immunofluorescence, the expression of IgG and complements was negative; however, IgA was positively expressed in a granular pattern along the GBM. An IgA subclass analysis revealed a significant deposition of IgA1-lambda (IgA1-λ). Electron microscopy revealed irregular and small non-organized and non-Randall-type granular electron-dense deposits in the GBM that were shaped like snow leopard spots.ConclusionsAfter corticosteroid therapy was initiated, the patient’s eosinophilia remarkably improved and his serum creatinine, IgG, and IgG4 levels decreased to within the normal ranges. However, massive proteinuria persisted. To our knowledge, this is the first reported case of IgG4-related TIN associated with IgA1-λ-type MIDD with membranous features.

Uromodulin-related autosomal-dominant tubulointerstitial kidney disease-pathogenetic insights based on a case.

Uromodulin-related autosomal-dominant tubulointerstitial kidney disease (ADTKD-UMOD) is a rare monogenic disorder that is characterized by tubulointerstitial fibrosis and progression of kidney function loss, and may progress to end-stage renal disease. It is usually accompanied by hyperuricaemia and gout. Mutations in the uromodulin gene (UMOD) resulting in malfunctioning of UMOD are known to be the cause of ADTKD-UMOD, which is assumed to be an endoplasmatic reticulum (ER) storage disease. As a case vignette, we report a 29-year-old female with a suspicious family history of chronic kidney disease presenting with progressive loss of renal function, hyperuricaemia and frequent urinary tract infections. Urinary tract infections and pyelonephritides may represent a clinical feature of uromodulin malfunction as it plays a protective role against urinary tract infections despite only sporadic data on this topic. ADTKD-UMOD was diagnosed after genetic testing revealing a missense mutation in the UMOD gene. Light microscopy showed excessive tubular interstitial fibrosis and tubular atrophy together with signs of glomerular sclerosis. Electron microscopic findings could identify electron dense storage deposits in the ER of tubular epithelial cells of the thick ascending loop. Immunohistological staining with KDEL (lysine, aspartic acid, glutamic acid, leucine) showed positivity in the tubular cells, which likely represents ER expansion upon accumulation of misfolded UMOD which could trigger the unfolded protein response and ER stress. This review highlights pathophysiological mechanisms that are subject to ADTKD-UMOD.

Unraveling the Molecular Mechanisms Underlying Complement Dysregulation by Nephritic Factors in C3G and IC-MPGN.

Membranoproliferative glomerulonephritis (MPGN) was recently classified as C3 glomerulopathies (C3G), and immune-complex (IC) mediated MPGN. Dysregulation of the complement alternative pathway, driven by acquired and/or genetic defects, plays a pathogenetic role in C3G. However, alternative pathway abnormalities were also found in IC-MPGN. The most common acquired drivers are the C3 nephritic factors (C3NeFs), heterogeneous autoantibodies that stabilize the C3 convertase, C3bBb. C3NeFs are traditionally detected by hemolytic assays based on sheep erythrocyte lysis, which however do not provide a direct molecular estimation of C3bBb formation and decay. We set up a microplate/western blot assay that specifically detects and quantifies C3bBb, and its precursor, the C3 proconvertase C3bB, to investigate the complex mechanistic effects of C3NeFs from patients with primary IC-MPGN (n = 13) and C3G (n = 13). In the absence of properdin, 9/26 patients had C3NeF IgGs stabilizing C3bBb against spontaneous and FH-accelerated decay. In the presence of properdin the IgGs of all but one patient had C3bBb-stabilizing activity. Properdin-independent C3NeFs were identified mostly in DDD patients, while properdin-dependent C3NeFs associated with either C3GN or IC-MPGN and with higher incidence of nephrotic syndrome. When we grouped patients based on our recent cluster analysis, patients in cluster 3, with highly electron-dense intramembranous deposits, low C3, and mostly normal sC5b-9 levels, had a higher prevalence of properdin-independent C3NeFs than patients in clusters 1 and 2. Conversely, about 70% of cluster 1 and 2 patients, with subendothelial, subepithelial, and mesangial deposits, low C3 levels and high sC5b-9 levels, had properdin-dependent C3NeFs. The flexibility of the assay allowed us to get deep insights into C3NeF mechanisms of action, showing that: (1) most C3NeFs bind strongly and irreversibly to C3 convertase; (2) C3NeFs and FH recognize different epitopes in C3 convertase; (3) C3NeFs bind rapidly to C3 convertase and antagonize the decay accelerating activity of FH on newly formed complexes; (4) C3NeFs do not affect formation and stability of the C3 proconvertase. Thus, our study provides a molecular approach to detecting and characterizing C3NeFs. The results highlight different mechanisms of complement dysregulation resulting in different complement profiles and patterns of glomerular injury, and this may have therapeutic implications.