NELL1 is a target antigen in malignancy-associated membranous nephropathy.

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.

Neural cell adhesion molecule 1 is a novel autoantigen in membranous lupus nephritis.

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.

Detection of SARS-CoV-2 in formalin-fixed paraffin-embedded tissue sections using commercially available reagents.

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.

Serum amyloid P deposition is a sensitive and specific feature of membranous-like glomerulopathy with masked IgG kappa deposits.

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.

Tissue staining for THSD7A in glomeruli correlates with serum antibodies in primary membranous nephropathy: a clinicopathological study.

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 A2 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.

An integrated pathology and ultrasonography-based simulation for training in performing kidney biopsy
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BACKGROUND: Medical practice trends and limitations in trainees' duty hours have diminished the interest and exposure of nephrology fellows to percutaneous kidney biopsy (PKB). We hypothesized that an integrated nephrology-pathology-led simulation may be an effective educational tool. MATERIALS AND METHODS: A 4-hour PKB simulation workshop (KBSW), led by two ultrasonography (US)-trained nephrologists and two nephropathologists, consisted of 6 stations: 1) diagnostic kidney US with live patients, 2) kidney pathology with plasticine models of embedded torso cross-sections, 3) US-based PKB with mannequin (Blue Phantom™), 4) kidney pathology with dissected cadavers, 5) US-based PKB in lightly-embalmed cadavers, and 6) tissue retrieval adequacy examination by microscope. A 10-question survey assessing knowledge acquisition and procedural confidence gain was administered pre- and post-KBSW. RESULTS: 21 participants attended the KBSW and completed the surveys. The overall percentage of correct answers to knowledge questions increased from 55 to 83% (p = 0.016). The number of "extremely confident" answers increased from 0 - 5% to 19 - 28% in all 4 questions (p = 0.02 - 0.04), and the number of "not at all confident" answers significantly decreased from 14 - 62% to 0 - 5% in 3 out of 4 questions (p = 0.0001 - 0.03). Impact of the imparted training on subsequent practice pattern was not assessed. CONCLUSION: A novel KBSW is an effective educational tool to acquire proficiency in PKB performance and could help regain interest among trainees in performing PKBs.
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Endothelin-1 upregulation mediates aging-related cardiac fibrosis.

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). ß-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.

Phenazopyridine associated acute interstitial nephritis and review of literature.

Phenazopyridine is a urinary analgesic; commonly seen side-effects of this drug include, orange discoloration of urine, methemoglobinemia, yellowish skin discoloration, hepatitis and acute renal failure. Various case reports with phenazopyridine associated acute renal failure secondary to acute tubular necrosis have been reported in the literature. Acute kidney injury in these patients is caused by either direct injury to renal tubular epithelial cells or secondary to pigment induced nephropathy from hemolytic anemia. Hypoxic injury from phenazopyridine-induced methemoglobinemia has been well documented. We report a case of biopsy proven acute interstitial nephritis, associated with therapeutic doses of phenazopyridine without any evidence of methemoglobinemia or other mechanism of renal injury. Clinicians should be aware of the toxicity of this commonly used drug and should look closely for signs of renal insufficiency. Identifying and stopping the offending medication stays as the first step, but recent studies indicate that early steroid administration improves renal recovery, as well as decreasing the risk of progression to chronic kidney disease with fibrosis and consequent permanent renal damage.

Smartphone-based machine learning model for real-time assessment of medical kidney biopsy.

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

Renal cortical infarction following treatment with sumatriptan in a kidney allograft recipient.

Renal cortical infarction is a rare cause of acute kidney injury that results from inadequate blood flow to the kidney, most commonly as a consequence of thrombotic or embolic occlusion of the renal artery or profound hypoperfusion. We report the case of a 78-year-old female kidney transplant recipient who developed a migraine headache, took sumatriptan, and soon after developed pain over the allograft and oligoanuric acute kidney injury. Kidney allograft biopsy showed renal cortical infarction. The mechanism of action of sumatriptan involves vasoconstriction, which counters the vasodilatation that is central to the pathogenesis of migraines. This case raises important questions regarding the safety of triptans with calcineurin inhibitors (which also act to vasoconstrict), particularly in elderly patients.

IgG4-related tubulointerstitial nephritis with plasma cell-rich renal arteritis.

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.

Acute kidney injury secondary to exposure to insecticides used for bedbug (Cimex lectularis) control.

Bedbug (Cimex lectularis) infestation is becoming a worldwide epidemic due to the emergence of insecticide-resistant strains. Pyrethroids are approved by the US Environmental Protection Agency for use against bedbugs and are considered minimally toxic to humans, with known respiratory, neurologic, and gastrointestinal effects. We present the first reported case of pyrethroid-induced toxic acute tubular necrosis (ATN). A 66-year-old healthy woman receiving no prior nephrotoxic medications presented with extreme weakness, decreased urine output, and acute kidney injury. She had administered multiple applications of a bedbug spray (permethrin) and a fogger (pyrethrin), exceeding the manufacturer's recommended amounts. She was found to have severe nonoliguric acute kidney injury associated with profound hypokalemia. Kidney biopsy revealed toxic ATN with extensive tubular degenerative changes and cytoplasmic vacuolization. With conservative management, serum creatinine level decreased from 13.0 mg/dL (estimated glomerular filtration rate, 3 mL/min/1.73 m(2)) to 1.67 mg/dL (estimated glomerular filtration rate, 37 mL/min/1.73 m(2)) within 6 weeks. Literature review uncovered no prior report of pyrethroid insecticide-induced ATN in humans, although there are reports of ATN with similar tubular vacuolization in rats exposed to this agent. Bedbug insecticides containing pyrethroids should be used with caution due to the potential development of toxic ATN after prolonged exposure.

BK polyoma virus nephropathy in the native kidney.

BACKGROUND: While BK polyoma virus nephropathy (PVN) is a well-recognized cause of renal allograft dysfunction, PVN of native kidneys is likely under-recognized. METHODS: We present the pathologic features, risk factors and outcomes of eight cases of PVN in native kidneys. RESULTS: The cohort included eight males aged 16-73 years (mean 47.4) with an immunocompromised state (mean duration 3.15 years) attributable to: hematologic malignancies (n = 6), for which three had undergone bone marrow transplant; lung transplant (n = 1) and combined tuberculosis and diabetes (n = 1). Seven patients were receiving specific immunosuppressive therapies. Patients were biopsied for acute kidney injury (AKI) with rise in mean creatinine levels from baseline 1.6 to 2.8 mg/dL. Pathology showed BK PVN with characteristic intranuclear inclusions staining positive for SV40 T antigen and negative for JC virus (JCV), with positive serum and/or urine PCR for BK virus. One patient had focal medullary JCV co-infection. Two patients also had renal infiltration by chronic lymphocytic leukemia (CLL). Six patients received specific therapy directed to PVN (cidofovir or leflunomide). Follow-up ranged from 2 to 20 (mean 10) months. Despite marked decrease in serum BK viral copy numbers, creatinine continued to rise in six cases (mean 3.7 mg/dL in four, requiring dialysis in two) and three patients died of malignancy, opportunistic infection or renal failure. Advanced histologic stage of PVN, ineffective antiviral therapy, co-morbidities and persistent immunocompromised state likely contributed to the poor outcomes. CONCLUSION: A high level of suspicion in immunocompromised patients is needed to diagnose PVN in an early stage that may respond more favorably to antiviral therapy.

Intrapancreatic accessory spleen: investigative dilemmas and role of EUS-guided FNA for diagnostic confirmation.

CONTEXT: We submit a case of intrapancreatic accessory spleen. CASE REPORT: A 33-year-old patient with history of dyspepsia underwent imaging studies suggestive of a neuroendocrine tumor. After referral to our institute, endoscopic ultrasound guided fine needle aspiration (EUS-FNA) confirmed diagnosis as intrapancreatic accessory spleen. DISCUSSION: An accessory spleen may develop from estranged mesenchymal cells due to fusion failure of the splenic anlage. The prevalence of an accessory spleen is 10-30% with 80% of them present at the splenic hilum and 17% in the pancreatic tail. Intrapancreatic accessory spleen is commonly misdiagnosed as a pancreatic tumor. Since, the differential diagnosis includes pancreatic neuroendocrine tumors, additional investigation with EUS-FNA should be considered when radiological diagnosis is not definitive. CONCLUSION: For diagnosis of intrapancreatic accessory spleen, radiographic imaging is useful, but lacks specificity without tissue diagnosis. Diagnosis can be safely and reliably established with EUS-FNA, leading to a benign prognosis and avoidance of unnecessary surgical intervention.

The significance of foot process effacement in immunoglobulin a nephropathy: clinicopathologic study of 161 cases with light, immunofluorescence and electron microscopic studies.

IGA nephropathy (IGAN) is the most common glomerular disease worldwide. Patients may present with hematuria and non-nephrotic (NNRP) or uncommonly nephrotic range proteinuria (NRP). To the authors' knowledge, correlation of podocyte foot process effacement (FPE) with subclasses of IGAN and proteinuria (PT) has not been studied. Retrospectively, 161 cases of IGAN with light, immunofluorescence, and electron microscopy (EM) were reviewed and classified according to Haas classification. EM was available in 110 out of 161 (67%) cases. FPE was evaluated as mild, <30%; moderate, 30–70%; and severe, >70% and was correlated with class and the level of PT. Out of 161 cases, 101 were males and 60 were females with M:F ratio of 1.71:1. In 72 cases, race was known as follows: white, 63 (88%); black, 6 (8%); Hispanic, 2 (3%); Asian, 1 (1%). Clinical history was available in 94 cases: PT 39 cases (42%), PT+hematuria 33 cases (35%), hematuria 15 cases (16%), and renal failure in 7 cases (7%). In 88 cases with FPE, PT was nephrotic in 21 and non-nephrotic in 29 cases. FPE is common in IGAN. No correlation between FPE and IGAN subclass (p=.42) or proteinuria group and IGAN subclass (p=.10) is present. Whether FPE is simply a reflection of other pathologic mechanisms and its significance in the pathophysiology of IGAN requires further investigation.

Cardiac hypertrophy during hypercholesterolemia and its amelioration with rosuvastatin and amlodipine.

Hypercholesterolemia is a common accompaniment of atherosclerosis and may be associated with cardiac hypertrophy. To define the mechanistic basis of cardiac hypertrophy in hypercholesterolemia, we fed low-density lipoprotein receptor knockout (LDLR KO) mice regular diet or high cholesterol (HC) diet for 26 weeks. There was clear evidence of cardiomyocyte hypertrophy and collagen deposition in the hearts of LDLR KO mice fed with HC diet, confirmed by histopathology (hematoxylin and eosin and Picrosirius staining) and upregulation of genes for brain natriuretic peptide, alpha-tubulin, transforming growth factor beta1, and connective tissue growth factor (CTGF). These changes were independent of change in blood pressure. The hypercholesterolemic mice hearts showed an upregulation of LOX-1, an oxidized low-density lipoprotein receptor, and angiotensin II type 1 receptor (AT1R) at messenger RNA level. In addition, there was a marked upregulation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor kappaB (NF-kappaB) messenger RNA, indicating overexpression of markers of oxidant stress. A separate group of LDLR KO mice were fed HC diet along with a potent 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitor rosuvastatin or a dihydropyridine calcium channel blocker amlodipine. Administration of rosuvastatin or amlodipine reduced the overexpression of genes for LOX-1 and AT1R and associated NADPH oxidase and NF-kappaB. These phenomena were associated with a marked decrease in cardiomyocyte hypertrophy and collagen deposits in and around the cardiomyocytes. In conclusion, this study provides evidence of cardiac hypertrophy and fibrosis in hypercholesterolemia independent of blood pressure change LOX-1 and AT1R act as possible signals for oxidant stress leading to alterations in cardiac structure during hypercholesterolemia. Most importantly, rosuvastatin and amlodipine ameliorate cardiomyocyte hypertrophy and fibrosis.

Proflavine Hemisulfate as a Fluorescent Contrast Agent for Point-of-Care Cytology.

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.