Parietal epithelial cells maintain the epithelial cell continuum forming Bowman's space in focal segmental glomerulosclerosis

In the glomerulus, Bowman's space is formed by a continuum of glomerular epithelial cells. In focal segmental glomerulosclerosis (FSGS), glomeruli show segmental scarring, a result of activated PECs invading the glomerular tuft. The segmental scars interrupt the epithelial continuum. However, non-sclerotic segments seem to be preserved even in glomeruli with advanced lesions. We studied the histology of the segmental pattern in Munich Wistar Frömter (MWF) rats, a model for secondary FSGS. Our results showed that matrix layers lined with PECs cover the sclerotic lesions. These PECs formed contacts with podocytes of the uninvolved tuft segments, restoring the epithelial continuum. Formed Bowman's spaces were still connected to the tubular system. Furthermore, in biopsies of patients with secondary FSGS we also detected matrix layers formed by PECs, separating the uninvolved from the sclerotic glomerular segments. While PECs have a major role in the formation of glomerulosclerosis, we showed that in FSGS, PECs also restore the glomerular epithelial cell continuum that surrounds Bowman's space. This process may be beneficial and indispensable for glomerular filtration in the uninvolved segments of sclerotic glomeruli.

Explainable Biomarkers for Automated Glomerular and Patient-Level Disease Classification

Background Pathologists use multiple microscopy modalities to assess renal biopsies. Besides usual diagnostic features, some changes are too subtle to be properly defined. Computational approaches have the potential to systematically quantitate subvisual clues, provide pathogenetic insight, and link to clinical outcomes. To this end, a proof of principle study is presented demonstrating that explainable biomarkers through machine learning can distinguish between glomerular disorders at the light microscopy level. Methods The proposed system employed image analysis techniques and extracted 233 explainable biomarkers related to color, morphology, and microstructural texture. Traditional machine learning was then used to classify minimal change disease (MCD), membranous nephropathy (MN), and thin-basement membrane nephropathy (TBMN) diseases on a glomerular and patient-level basis. Results The final model combined the Gini feature importance set and Linear Discriminant Analysis classifier. Six morphological (nuclei-to-glomerular tuft area, nuclei-to-glomerular area, glomerular tuft thickness > 10, glomerular tuft thickness > 3, total glomerular tuft thickness, and glomerular circularity) and four microstructural texture features (luminal contrast using wavelets, nuclei energy using wavelets, nuclei variance using color vector LBP, and glomerular correlation using GLCM) were together the best performing biomarkers. Accuracies of 76.86% and 86.67% were obtained for glomerular and patient-level classification, respectively. Conclusion Computational methods using explainable glomerular biomarkers have diagnostic value and are compatible with our existing knowledge of disease pathogenesis. Furthermore, this algorithm can be applied to clinical datasets for novel prognostic and mechanistic biomarker discovery.

MO1030THE FIRST CASE OF ATYPICAL HEMOLYTIC UREMIC SYNDROME (AHUS) FEATURES IN AN EXPERIMENTAL MODEL

Background and Aims Thrombotic Microangiopathies (TM) are three distinct clinical syndromes presenting the same histological renal pattern: typical and atypical Hemolytic Uremic Syndrome (SEU- aSEU) and Thrombotic Thrombocytopenic Purpura (TTP). So far, the first report of a TM has been generally attributed to Eli Moschowitz. In 1922 he described the case of a 16 years old girl who died after acute onset of fever with petechial lesions and autoptic findings of hyaline thrombosis of terminal arterioles and capillaries, thus profiling the first case of TTP. Only in the 1955, Conrad Gasser described the first medical record of HUS, describing the case of a patient with a manifestation of bilateral necrosis of the renal cortex. We describe the first reported case of a Thrombotic Microangiopathy, in particular an experimentally induced aHUS by Richard M. Pearce in 1909. In this case, the trigger leading to aHUS was represented by snake venom injection in an experimental rabbit model. Method Pearce described acute glomerular lesions produced in the rabbit using dried venom of rattlesnake Crotalus Adamanteus. It was dissolved in salt solution in the proportion of 0.25 of a milligram to one cubic centimeter, rising gradually to two milligrams, and then followed by doses of 0.5 of a milligram of fresh venom at various intervals. The intervals between injections depended on the general condition of the animal and the amount of albuminuria. Results Rabbit kidneys showed well marked hemorrhagic and exudative lesions in the glomeruli; hyaline, granular, blood, and hemoglobin casts in both convoluted and collecting tubules; and granular degeneration of the epithelium of the convoluted tubules and loops of Henle. Pearce also described a “penetration of the cells of the compressed glomerular tuft into the mass of hemorrhage lying either in the tuft itself or in the capsular space” (Figure 1). In animals surviving 20-30 days after the first injection of the venom, the acute lesions were demonstrated to subside at the microscopic examination and their earlier presence was marked by “occasional casts and compressed masses of red cells in the glomerular spaces and tufts”. At the same time other models also showed “extensive granular degeneration of the convoluted tubules and many casts”. The renal autoptic findings presented the features of a vascular nephritis with severe endothelial changes. Conclusion Glomerular and tubular lesions of rabbits’ kidneys induced by crotalus’s venom showed typical features of what is today defined as aHUS. In this first experiment, the author described a glomerular tuft as “more analogous to the organization of a red thrombus than it is to any form of glomerular lesion known in man”, so we can affirm that Pearce described, ante litteram TMA histological features many years before other scientists.

MO087PLASMALEMMAL VESICLE-ASSOCIATED PROTEIN-1 (PLVAP) INDICATES THE FORMATION OF DIAPHRAGM-BRIDGED FENESTRATIONS OF GLOMERULAR ENDOTHELIAL CELLS IN KIDNEY DISEASE

Background and Aims Plasmalemmal vesicle-associated protein-1 (PLVAP or PV-1) is a major protein of diaphragm-bridged fenestrated endothelial cells found in capillaries of neuroendocrine glands and peritubular capillaries. In contrast to peritubular capillaries, the glomerulus is known for its unique fenestrated endothelium without any diaphragm formation thereby ensuring free filtration. Here we aimed to investigate whether PLVAP is expressed in glomerular endothelial cells in various glomerular diseases and whether PLVAP expression is associated with the formation of diaphragm-bridged endothelial cells. Method A total number of 114 biopsy samples of glomerular diseases including diabetic nephropathy, FSGS, IgA-Nephritis, ANCA-GN and Lupus–Nephritis were analyzed immunohistochemistically for glomerular PLVAP expression. A fraction of PLVAP positive cases was subsequently investigated ultrastrucurally for the formation of diaphragm-bridged glomerular endothelial cells. Results One third of all cases showed at least one glomerulus with one single circumferential PLVAP staining. Interestingly, the most prominent staining, affecting the entire glomerular tuft, was observed in diabetic nephropathy and ANCA-GN. Ultrastructurally, such cases exhibited injured endothelium with focal detachment from the glomerular basement membrane, loss of pore formation and frequently diaphragm-bridged fenestrations reminiscent of peritubular capillaries. Conclusion Our data show that injured glomerular endothelium is capable of forming true diaphragm-bridged fenestrations, suggesting a possible role in preventing glomerular protein leakage and limiting its detachment from the GBM.

The Nephroprotective Effects of Moringa Oleifera Extract against Contrast Induced Nephrotoxicity

Objectives: The present research has been conducted to assess the Nephroprotective effects of the Moringa Oleifera Extract against contrast -induced nephrotoxicity in male rabbits. Study Design: Experimental design Place and Duration of Study: it was carried out in the Faculty of Pharmacy/University of Kufa, Iraq/Al-Najaf Town .The study began at October 2020 and end at March 2021. Methodology: Twenty one adult (male rabbits) weighing range(1.2-1.5kg). Rabbits are divided in a random manner into three equal groups each group including seven rabbits. Group1:control group administered DW, Group2:has been treated with Iodide contrast(2.5mg/kg) of (370mg/ml),group 3: dosed alcoholic extracts of Moringa 250 mg/kg and contrast (2.5mg/kg).By accounting data on the MDA levels ,Glutathione level and features of kidney histopathology. All efforts have been made to reduce the suffering of a animals prior to and throughout the experiment and sampling. Results: The alcoholic Moringa extract at 250mg/kg body weight doses orally considerably protected contrast induced toxicity of the kidneys in the rabbits through the increment of the level of GSH and reduces the level of MDA. In the Histological data, the extract activities also protect the damage of the kidney, which is induced by the contrast that has been represented by the de-generation of some tubules and atrophy of glomerular, glomerular tuft congestion and expanded Bowman’s Space. Conclusions: This study proposed that Moringa’s alcoholic extract results in the enhancement of the defense status of the oxidative stress against the renal toxicity.(Moringa Oleifera have Nephroprotective effect against contrast induce Nephrotoxicity).

Endothelial-Specific Deletion of CD146 Protects Against Experimental Glomerulonephritis in Mice

CD146 is an endothelial junctional adhesion molecule, which expression is increased in human glomerular diseases. However, the pathological significance of this overexpression remains unknown. Induction of glomerulonephritis in mice, by using nephrotoxic serum, showed that CD146 expression was highly induced within damaged glomeruli and was associated with renal inflammation and fibrosis. Interestingly, 2 weeks after glomerulonephritis induction, CD146 knockout mice showed preserved renal function as proteinuria and blood urea nitrogen levels were significantly lower compared with wild-type littermates. Furthermore, renal structure was considerably conserved, since crescents formation, tubular dilation, monocyte and lymphocyte infiltration, and interstitial renal fibrosis were highly reduced. Colocalization with markers for different types of glomerular cells showed that CD146 expression was mainly increased within the injured endothelium of the glomerular tuft. Consequently, we generated a new transgenic strain in which CD146 was specifically deleted in the vascular endothelium. Similarly to CD146 knockout, these mice showed preservation of renal structure and function after the induction of glomerulonephritis compared with wild-type animals. These data show that endothelial CD146 plays a major role in glomerulonephritis and may represent a novel therapeutic target to reduce glomerular damage and the progression of renal disease.

Toxicity studies of sea anemone (Gyrostoma helianthus) crude extract and fractionated proteins on liver and kidney in rats

Aim: To study the poisonous effects of crude extract and fractionated protein of sea anemone Gyrostoma helianthus on histological and biochemical parameters in male rats. Methodology: Live specimens of Gyrostoma helianthus were collected from Jeddah coast, fixed in ethanol, dried and powdered to obtain the crude residue extract. Some of the crude extract were ultra-filtered to produce protein fractions of 1 and 3KDa. The LD50 of crude extract powder was estimated for male rats and momentary repeated subacute dosing (1/4 LD50) for 7 days was carried out to obtain toxicity data. Histological examination of liver and kidney sections were carried out. Serum whole protein, total albumin, ALT and LDH were estimated by standard protocol. Results: The LD50 of crude extract was 20.32 mg kg-1 for male rats. In acute and subsequent sub-acute toxicity, neurological symptoms such as convulsions, paralysis, tremors, and ataxia were observed overdose exposure. At the end of exposure to subacute dose histopathological changes like hemolyzed blood and atrophy of glomerular tuft in kidney and fatty changes, vacuolation, necrosis, and infiltration in liver was noted. Furthermore, vital significant increase in total protein, ALT and LDH and reduced bilirubin in serum of treated groups was observed as compared to the control. Interpretation: The present study emphasizes the toxicological, behavioral, biochemical, and histological bioactivity of crude extract and protein fractions of 1 and 3 KDa of Gyrostoma helianthus sea anemone, which is commonly found in the Red Sea. The tested extracts were found to be active at a concentration 5.08 mg kg-1. The yielded effects may interpret treatment strategies of toxicological and pharmacologic intervention. Key words: Biochemistry, Gyrostoma helianthus, Histopathology, Toxicity

DIAGNOSIS OF MYXOBOLUS BRAMAE (MYXOSPOREA: MYXOBOLIDAE) IN THE KIDNEYS TISSUE OF CARASOBARBUS LUTEUS AND HISTOPATHOLOGICAL CHANGES ASSOCIATED WITH INFECTION

This study was conducted during the period from March till the end of October 2018, to study the histopathological changes of Myxobolus bramae in kidney tissue of Carasobarbus luteus caught from Tigris River passing through Baghdad city. During the period of this study, a total of 60 fishes belonging to Carasobarbus luteus species from the family Cyprinidae were collected. The prevalence of infection with these protozoa was determinate (5.00%). Histopathological study due to M. bramae in the kidney tissue of C. luteus was done by using three types of stain: Hemotoxylin and eosin, giemsa and acid fast stain to observe plasmodia cyst and structures of spores. These changes characterized by tubular degeneration, necrosis, hyalinization of glomerular tuft, mild distension of Bowman᾿s space with a reduction in haemopoitic tissue together with inflammatory response, and accumulation of melanomacrophages at the site of infection. The results of this study revealed that Carasobarbus luteus from Tigris River at Baghdad city, it infected with Myxobolus bramae and this parasite cause severe histopathological changes in the infected kidneys tissue.

P0420PARTIAL PHARMACOGENETIC PODOCYTE DEPLETION IN A HIGH-THROUGHPUT ZEBRAFISH MODEL RESEMBLES HUMAN FOCAL AND SEGMENTAL GLOMERULOSCLEROSIS

Background and Aims Although focal and segmental glomerulosclerosis (FSGS) has been in the scientific focus for many years, it is still a massive burden for patients with no causal therapeutic option. In FSGS, glomerular podocytes are injured, parietal epithelial cells (PECs) are activated and engage in the formation of cellular lesions leading to progressive glomerular scarring. Therefore, novel drug-screening assays are needed. Unfortunately, simple cellular in vitro-based screening assays are not ideal as glomerular architecture and crosstalk between glomerular cells is insufficiently modelled. Therefore, reliable animal models are still required for drug development, which unfortunately are not ideal for high-throughput applications. To date, due to its size, easy maintenance and breeding, zebrafish larvae are the simplest vertebrate model that are used in high-content screenings. Until today, it was unclear whether zebrafish can be used as a model for human FSGS. We therefore aimed to investigate whether partial podocyte-depletion in larval zebrafish leads to formation of FSGS-like disease and if the model can be used for screening purposes. Method We used a transgenic zebrafish model of pharmacogenetic podocyte depletion: In the Tg(nphs2:GAL4), Tg(UAS:Eco.nfsb-mCherry) strain, podocytes express the bacterial nitroreductase under control of the podocin promotor and can be dose-dependently ablated upon administration of metronidazole. Proteinuria was quantified using in vivo confocal laser scanning microscopy of intravenously administered high-molecular-weight fluorescent dextran. Plastic-embedded larvae where histologically and morphometrically assessed using HE, PAS and Jone’s silver staining after metronidazole washout. Glomerular ultrastructure was assessed using transmission electron microscopy of ultrathin sections. Immunofluorescence staining was carried out on kryosections to investigate extracellular matrix deposition (collagen-1, laminin), cellular proliferation (pcna) as well as parietal cell origin and activation (pax2a). Results To partially deplete podocytes, larvae where treated with 80 µM metronidazole from 4-6 days post fertilization, so that a subset of podocytes was depleted. In contrast to controls, podocyte-depleted larvae developed severe whole-body edema (Fig. A). Dynamic in vivo imaging of intravascular 500 kDa fluorescent dextran revealed massive leakage of the glomerular filtration barrier. Ultrastructural and immunofluorescent evaluation showed broad foot process effacement of remaining podocytes (Fig. D) and massive decrease of the slit diaphragm component podocin. Moreover, we found numerous sub-podocyte space pseudocysts (asterisk in Fig. D), microvillous transformation and formation of podocytic tight junctions as well as parietovisceral adhesions of the two layers of Bowman’s capsule. Parietal epithelial cells where activated, changed their phenotype towards a cuboidal shape, began to proliferate as demonstrated by pcna immunofluorescence and where recruited to cellular lesions on the glomerular tuft as demonstrated by the presence of cuboidal pax2a+ cells on the glomerular tuft (arrowheads Fig. B). Moreover, we found significant extracellular matrix deposition by the pax2a+ cells as demonstrated by Jone’s silver staining and laminin immunofluorescence (Fig. C). Conclusion Herein we show that upon podocyte-depletion, zebrafish larvae develop important functional and morphological features of human FSGS such as severe proteinuria and edema, podocyte foot process effacement, activation of parietal epithelial cells which contribute to cellular lesions and deposit extracellular matrix on the glomerular tuft. We conclude that this model resembles the human disease in important features and therefore propose its applicability for a high-throughput drug screening assay for FSGS.