Patients with membranous nephropathy have an increased risk of malignancy compared to the general population, but the target antigen for malignancy-associated membranous nephropathy is unknown. To ...explore this, we utilized mass spectrometry for antigen discovery in malignancy-associated membranous nephropathy examining immune complexes eluted from frozen kidney biopsy tissue using protein G bead immunoglobulin capture. Antigen discovery was performed comparing cases of membranous nephropathy of unknown and known type. Mass spectrophotometric analysis revealed that nerve epidermal growth factor-like 1 (NELL1) immune complexes were uniquely present within the biopsy tissue in membranous nephropathy. Additional NELL1-positive cases were subsequently identified by immunofluorescence. In a consecutive series, 3.8% of PLA2R- and THSD7A-negative cases were NELL1-positive. These NELL1-positive cases had segmental to incomplete IgG capillary loop staining (93.4%) and dominant or co-dominant IgG1-subclass staining (95.5%). The mean age of patients with NELL1-positive membranous nephropathy was 66.8 years, with a slight male predominance (58.2%) and 33% had concurrent malignancy. Compared with PLA2R- and THSD7A-positive cases of membranous nephropathy, there was a greater proportion of cases with malignancies in the NELL1-associated group. Thus, NELL1-associated membranous nephropathy has a unique histopathology characterized by incomplete capillary loop staining, IgG1-predominance, and is more often associated with malignancy than other known types of membranous nephropathy.
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Membranous lupus nephritis is a frequent cause of nephrotic syndrome in patients with systemic lupus erythematosus. It has been shown in phospholipase A2 receptor positive membranous nephropathy that ...known antibodies can be detected within sera, determination of the target autoantigen can have diagnostic significance, inform prognosis, and enable non-invasive monitoring of disease activity. Here we utilized mass spectrometry for antigen discovery in laser captured microdissected glomeruli from formalin-fixed paraffin embedded tissue and tissue protein G immunoprecipitation studies to interrogate immune complexes from frozen kidney biopsy tissue. We identified neural cell adhesion molecule 1 (NCAM1) to be a target antigen in some cases of membranous lupus nephritis and within rare cases of primary membranous nephropathy. The prevalence of NCAM1 association was 6.6% of cases of membranous lupus nephritis and in 2.0% of primary membranous nephropathy cases. NCAM1 was found to colocalize with IgG within glomerular immune deposits by confocal microscopy. Additionally, serum from patients with NCAM1-associated membranous nephropathy showed reactivity to NCAM1 recombinant protein on Western blotting and by indirect immunofluorescence assay, demonstrating the presence of circulating antibodies. Thus, we propose that NCAM1 is a target autoantigen in a subset of patients with membranous lupus nephritis. Future studies are needed to determine whether anti-NCAM1 antibody levels correlate with disease activity or response to therapy.
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Renal biopsies performed in diabetic patients are increasing in number and complexity. This study sought to determine the usefulness of renal biopsy in patients with diabetes and the predictability ...of diagnosing diabetic nephropathy (DN) versus nondiabetic renal disease (NDRD) from clinical and laboratory data.
To assess modern trends, a retrospective study was performed of clinical-pathologic findings in all patients with diabetes who had a biopsy in 2011. Among 2642 native kidney biopsies, 620 (23.5%) were from patients with diabetes.
The cohort included 371 men (60.7%) aged a median (interquartile range) 62 years (52-69) with 10-year (5-15) duration of diabetes mellitus (DM). Median serum creatinine was 2.5 mg/dl (1.6-4.4), and 52% of patients had stage 4-5 CKD. On biopsy, 37% of patients had DN alone, 36% had NDRD alone, and 27% had DN plus NDRD. In NDRD alone, FSGS (22%), hypertensive nephrosclerosis (18%), acute tubular necrosis (ATN) (17%), IgA nephropathy (11%), membranous GN (8%), and pauci-immune GN (7%) comprised 80% of diagnoses, compared with ATN (43%), hypertensive nephrosclerosis (19%), FSGS (13%), and IgA nephropathy (7%) for DN plus NDRD. In multivariate analyses, longer duration of DM was associated with a greater likelihood of DN and a lower likelihood of NDRD: each added year of DM reduced the odds of NDRD by 5% (odds ratio, 0.95; 95% confidence interval, 0.91 to 0.98; P=0.004). DM duration ≥ 12 years was the best predictor (58% sensitivity, 73% specificity) of DN alone.
Approximately one-quarter of all renal biopsies are performed in patients with DM. Judicious use of renal biopsy has uncovered NDRD alone or superimposed on DN in the majority of such biopsies. ATN is emerging as an important category of NDRD, which has not been reported previously.
Coronavirus Disease-19 (COVID-19), caused by the coronavirus SARS-CoV-2, was initially recognized in Wuhan, China and subsequently spread to all continents. The disease primarily affects the lower ...respiratory system, but may involve other organs and systems. Histopathologic evaluation of tissue from affected patients is crucial for diagnostic purposes, but also for advancing our understanding of the disease. For that reason, we developed immunohistochemical (IHC) and in situ hybridization (ISH) assays for detection of the. virus. A total of eight autopsy lungs, one placenta, and ten kidney biopsies from COVID-19 patients were stained with a panel of commercially available antibodies for IHC and commercially available RNA probes for ISH. Similarly, autopsy lungs, placentas and renal biopsies from non-COVID-19 patients were stained with the same antibodies and probes. All eight lungs and the placenta from COVID-19 patients stained positive by IHC and ISH, while the kidney biopsies stained negative by both methodologies. As expected, all specimens from non-COVID-19 patients were IHC and ISH negative. These two assays represent a sensitive and specific method for detecting the virus in tissue samples. We provide the protocols and the list of commercially available antibodies and probes for these assays, so they can be readily implemented in pathology laboratories and medical examiner offices for diagnostic and research purposes.
Detailed protocols for immunohistochemical and in situ hybridization assays for the detection of SARS-CoV-2 are provide so they can be readily implemented in pathology laboratories and medical examiner offices for diagnostic and research purposes. These assays were found to represent a sensitive and specific method for detecting the virus in tissue samples.
Thrombospondin type-1 domain-containing 7A (THSD7A) is the most recently recognized target antigen in patients with membranous nephropathy. We stained membranous nephropathy biopsies processed in our ...laboratory for phospholipase A
receptor and THSD7A over an 18-month period and selected all THSD7A-positive cases for study. Serum samples from most patients were tested by an indirect immunofluorescence assay for the presence of THSD7A antibodies (Euroimmun). A total of 31 patients were diagnosed with THSD7A-associated membranous nephropathy for a prevalence of 2.4% among patients with membranous nephropathy. The patients were most often male (male-to-female ratio of 1.6) with a mean age of 62 years and a mean proteinuria of 9.6 g per day (range 1.1-15.9). Two of the 31 patients had a history of cancer and none were diagnosed with malignancy on follow-up. Serum samples were available at the time of biopsy from 24 patients and all tested positive for antibodies against THSD7A. Conversely, all 20 serum samples from patients with membranous nephropathy who had negative staining for THSD7A were negative for serum reactivity to THSD7A. We conclude that THSD7A tissue staining of kidney biopsies with membranous nephropathy is a sensitive and specific method for the diagnosis of THSD7A-associated membranous nephropathy and it correlates strongly with the serum antibody testing. We also present the clinicopathologic details of the largest cohort to date of THSD7A-associated membranous nephropathy from a single institution.
Abstract Endothelin-1 (ET-1) plays a major role in regulating myocardial fibrosis in several pathological conditions, such as hypertension and diabetes. Aging is an independent risk factor for ...myocardial fibrosis. We hypothesized that ET-1 upregulation may be a basis of enhanced collagen synthesis in the senescent fibroblasts resulting in cardiac fibrosis with aging. To examine this hypothesis, we cultured mouse cardiac fibroblasts to passage-30 (P30). β–Galactosidase activity and several other aging markers were markedly increased in P30 (vs. P3) fibroblasts, indicating that these cells were indeed undergoing senescence. Importantly, ET-1 expression was markedly upregulated in P30 (vs. P3) fibroblasts. Of note, estrogen receptor-α (ER-α), an important negative regulator of ET-1, was downregulated in P30 fibroblasts. We also studied aged (130-weeks old, female) mice hearts, and observed that ET-1 was upregulated and ER-α was downregulated in these hearts (vs. 6-week old mice hearts, female). Similar observations were made in the fibroblasts isolated from aged mice hearts. ET-1 upregulation with aging was also seen in ≈ 70-year old (vs. ≈ 30-year old) human heart sections. In concert with ET-1 upregulation, the expression of fibronectin and collagens was found to be markedly increased in P30 cardiac fibroblasts in culture, fibroblasts isolated from the aged mice hearts, and in aged human hearts. Interestingly, inhibition of ET-1 in the senescent P30 fibroblasts by 2 different strategies (the use of siRNA and the use of endothelin converting enzyme inhibitors) markedly suppressed expression of fibrosis signals. Further, treatment with synthetic ET-1 enhanced fibronectin and collagen expression in P3 cardiac fibroblasts. These observations in mice and human hearts suggest that aging-related cardiac fibrosis is, at least partially, dependent on the upregulation of ET-1.
Kidney biopsy is the gold-standard for diagnosing medical renal diseases, but the accuracy of the diagnosis greatly depends on the quality of the biopsy specimen, particularly the amount of renal ...cortex obtained. Inadequate biopsies, characterized by insufficient cortex or predominant medulla, can lead to inconclusive or incorrect diagnoses, and repeat biopsy. Unfortunately, there has been a concerning increase in the rate of inadequate kidney biopsies, and not all medical centers have access to trained professionals who can assess biopsy adequacy in real time. In response to this challenge, we aimed to develop a machine learning model capable of assessing the percentage cortex of each biopsy pass using smartphone images of the kidney biopsy tissue at the time of biopsy.
747 kidney biopsy cores and corresponding smartphone macro images were collected from five unused deceased donor kidneys. Each core was imaged, formalin-fixed, sectioned, and stained with Periodic acid–Schiff (PAS) to determine cortex percentage. The fresh unfixed core images were captured using the macro camera on an iPhone 13 Pro. Two experienced renal pathologists independently reviewed the PAS-stained sections to determine the cortex percentage. For the purpose of this study, the biopsies with less than 30% cortex were labeled as inadequate, while those with 30% or more cortex were classified as adequate. The dataset was divided into training (n=643), validation (n=30), and test (n=74) sets. Preprocessing steps involved converting High-Efficiency Image Container iPhone format images to JPEG, normalization, and renal tissue segmentation using a U-Net deep learning model. Subsequently, a classification deep learning model was trained on the renal tissue region of interest and corresponding class label.
The deep learning model achieved an accuracy of 85% on the training data. On the independent test dataset, the model exhibited an accuracy of 81%. For inadequate samples in the test dataset, the model showed a sensitivity of 71%, suggesting its capability to identify cases with inadequate cortical representation. The area under the receiver-operating curve (AUC-ROC) on the test dataset was 0.80.
We successfully developed and tested a machine learning model for classifying smartphone images of kidney biopsies as either adequate or inadequate, based on the amount of cortex determined by expert renal pathologists. The model's promising results suggest its potential as a smartphone application to assist real-time assessment of kidney biopsy tissue, particularly in settings with limited access to trained personnel. Further refinements and validations are warranted to optimize the model's performance.
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•We developed a machine learning model to perform binary classification of smartphone images of kidney biopsy tissue, based on the amount of cortex.•The model was developed on smartphone images of 747 kidney biopsy cores from deceased donor kidneys, imaged using an iPhone 13 Pro.•Preprocessing involved image conversion, normalization, and renal tissue segmentation with U-Net.•On the independent test dataset, the model achieved an overall accuracy of 81%, and sensitivity of 71% for identifying samples containing less than 30% cortex.•The model can be deployed as a smartphone application to assist real-time assessment of renal biopsy cores at the time of biopsy, especially in the settings with limited access to trained personnel.
Membranous-like glomerulopathy with masked IgG kappa deposits (MGMID) is a recently described pattern of glomerulonephritis with a unique histopathology. The pattern is characterized by subepithelial ...and/or mesangial immune deposits that are “masked”, to immunoglobulin staining by routine immunofluorescence but strongly stain for IgG and kappa light chain after protease digestion. Patients with this pattern of glomerulonephritis are most commonly young females presenting with proteinuria and a vague history of autoimmune disease such as low titer antinuclear antibodies. Here we compared the mass spectrometry profile of laser capture microdissected glomeruli from nine MGMID renal biopsies with eight biopsies showing other patterns of membranous glomerulopathy. The protein most significantly increased in MGMID was serum amyloid P. Immunostaining showed serum amyloid P colocalized with IgG in the glomeruli of MGMID but not with PLA2R-associated membranous glomerulopathy. Serum amyloid P was positive in the glomeruli of all 32 MGMID biopsies but negative in biopsies of other types of membranous glomerulopathies such as those associated with PLA2R and THSD7A. There were four biopsies with glomerular serum amyloid P staining among the 173 biopsies that did not fulfill criteria for MGMID or amyloidosis. All four of these biopsies with positive serum amyloid P staining had a membranous pattern of glomerulopathy with IgG kappa deposits that only differed from MGMID by the lack of “masking”. Thus, positive staining within glomerular deposits for serum amyloid P identifies a unique form of glomerulonephritis likely sharing a common pathophysiologic mechanism of disease.
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Proflavine hemisulfate, an acridine-derived fluorescent dye, can be used as a rapid stain for cytologic examination of biological specimens. Proflavine fluorescently stains cell nuclei and ...cytoplasmic structures, owing to its small amphipathic structure and ability to intercalate DNA. In this manuscript, we demonstrated the use of proflavine as a rapid cytologic dye on a number of specimens, including normal exfoliated oral squamous cells, cultured human oral squamous carcinoma cells, and leukocytes derived from whole blood specimens using a custom-built, portable, LED-illuminated fluorescence microscope. No incubation time was needed after suspending cells in 0.01% (w/v) proflavine diluted in saline. Images of proflavine stained oral cells had clearly visible nuclei as well as granular cytoplasm, while stained leukocytes exhibited bright nuclei, and highlighted the multilobar nature of nuclei in neutrophils. We also demonstrated the utility of quantitative analysis of digital images of proflavine stained cells, which can be used to detect significant morphological differences between different cell types. Proflavine stained oral cells have well-defined nuclei and cell membranes which allowed for quantitative analysis of nuclear to cytoplasmic ratios, as well as image texture analysis to extract quantitative image features.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Immunoglobulin G4 (IgG4)-related tubulointerstitial nephritis is a newly recognized clinicopathologic entity that may occur as an isolated renal lesion or as part of a multisystem disorder. It is ...characterized by plasma cell–rich interstitial nephritis with abundant IgG4-positive plasma cells and IgG-dominant tubulointerstitial immune deposits. We report the first case of IgG4-related tubulointerstitial nephritis with multifocal plasma cell–rich renal arteritis presenting as acute kidney injury in a 72-year-old man. Seven weeks of prednisone therapy led to nearly complete recovery of kidney function. This case enlarges the morphologic spectrum of this disorder and emphasizes the need to distinguish it from other causes of renal vasculitis.
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