Basement Membrane Deposition Research Articles

Integrated bioinformatics analysis for identifying fibroblast-associated biomarkers and molecular subtypes in human membranous nephropathy

BackgroundMembranous nephropathy (MN) is characterized by immune complex deposition in the glomerular basement membrane, leading to proteinuria and potentially progressive renal dysfunction. Fibroblasts have been implicated in the pathogenesis of MN through their involvement in tissue remodeling and immune modulation. MethodsWe employed integrated bioinformatics analyses to identify fibroblast-associated biomarkers and molecular subtypes in MN. The xCell algorithm was used to assess fibroblast infiltration, and weighted gene co-expression network analysis (WGCNA) identified fibroblast-related gene modules. Differentially expressed fibroblast-associated genes (DEFAGs) were screened between MN and healthy controls (HC) using differential expression analysis and protein-protein interaction (PPI) network construction. Consensus clustering categorized MN patients into distinct subtypes based on DEFAG expression profiles. ResultsFibroblast scores were a significant elevation in MN compared to HC, indicating increased fibroblast involvement in MN pathogenesis. WGCNA identified 13 fibroblast-related gene modules, with the brown and turquoise modules showing strong correlation with fibroblast scores (correlation coefficient = 0.79 and 0.75, respectively, p < 0.01). DEFAG analysis revealed 308 genes overlapping between WGCNA and differentially expressed genes (DEGs) in MN. Consensus clustering identified two molecular subtypes (C1 and C2) based on DEFAG expression patterns, with differential gene expression enriching pathways related to immune response and extracellular matrix remodeling. Core biomarker analysis highlighted COL3A1 and TGFB1 as central genes associated with MN, with elevated expression validated across multiple datasets. ConclusionIntegrated bioinformatics analysis identified fibroblast-associated molecular subtypes in MN, revealing distinct immune profiles and biomarkers. COL3A1 emerged as a potential diagnostic and therapeutic target, implicating its role in immune regulation and disease progression in MN.

Obesity-driven changes in breast tissue exhibit a pro-angiogenic extracellular matrix signature

Obesity has reached epidemic proportions in the United States, emerging as a risk factor for the onset of breast cancer and a harbinger of unfavorable outcomes [1–3]. Despite limited understanding of the precise mechanisms, both obesity and breast cancer are associated with extracellular matrix (ECM) rewiring [4–6]. Utilizing total breast tissue proteomics, we analyzed normal-weight (18.5 to < 25 kg/m2), overweight (25 to < 30 kg/m2), and obese (≥30 kg/m2) individuals to identify potential ECM modifying proteins for cancer development and acceleration. Obese individuals exhibited substantial ECM alterations, marked by increased basement membrane deposition, angiogenic signatures, and ECM-modifying proteins. Notably, the collagen IV crosslinking enzyme peroxidasin (PXDN) emerged as a potential mediator of the ECM changes in individuals with an elevated body mass index (BMI), strongly correlating with angiogenic and basement membrane signatures. Furthermore, glycan-binding proteins galectin-1 (LGALS1) and galectin-3 (LGALS3), which play crucial roles in matrix interactions and angiogenesis, also strongly correlate with ECM modifications. In breast cancer, elevated PXDN, LGALS1, and LGALS3 correlate with reduced relapse-free and distant-metastatic-free survival. These proteins were significantly associated with mesenchymal stromal cell markers, indicating adipocytes and fibroblasts may be the primary contributors of the obesity-related ECM changes. Our findings unveil a pro-angiogenic ECM signature in obese breast tissue, offering potential targets to inhibit breast cancer development and progression.

Chemical chaperones to the rescue of Alport syndrome?

Alport syndrome is a hereditary kidney disease caused by collagen IV mutations that interfere with the formation and deposition of the α3α4α5 protomer into the glomerular basement membrane. In this issue, Yu etal. show that the chemical chaperone tauroursodeoxycholic acid prevented kidney structural changes and function decline in mice with a pathogenic missense Col4a3 mutation by increasing mutant α3α4α5 protomer glomerular basement membrane deposition and preventing podocyte apoptosis induced by endoplasmic reticulum stress.

Selective mural cell recruitment of pericytes to networks of assembling endothelial cell-lined tubes.

Mural cells are critically important for the development, maturation, and maintenance of the blood vasculature. Pericytes are predominantly observed in capillaries and venules, while vascular smooth muscle cells (VSMCs) are found in arterioles, arteries, and veins. In this study, we have investigated functional differences between human pericytes and human coronary artery smooth muscle cells (CASMCs) as a model VSMC type. We compared the ability of these two mural cells to invade three-dimensional (3D) collagen matrices, recruit to developing human endothelial cell (EC)-lined tubes in 3D matrices and induce vascular basement membrane matrix assembly around these tubes. Here, we show that pericytes selectively invade, recruit, and induce basement membrane deposition on EC tubes under defined conditions, while CASMCs fail to respond equivalently. Pericytes dramatically invade 3D collagen matrices in response to the EC-derived factors, platelet-derived growth factor (PDGF)-BB, PDGF-DD, and endothelin-1, while minimal invasion occurs with CASMCs. Furthermore, pericytes recruit to EC tube networks, and induce basement membrane deposition around assembling EC tubes (narrow and elongated tubes) when these cells are co-cultured. In contrast, CASMCs are markedly less able to perform these functions showing minimal recruitment, little to no basement membrane deposition, with wider and shorter tubes. Our new findings suggest that pericytes demonstrate much greater functional ability to invade 3D matrix environments, recruit to EC-lined tubes and induce vascular basement membrane matrix deposition in response to and in conjunction with ECs.

Prickle1-driven basement membrane deposition of the iPSC-derived embryoid bodies is separable from the establishment of apicobasal polarity.

Basement membrane (BM) component deposition is closely linked to the establishment of cell polarity. Previously, we showed that Prickle1 is crucial for BM deposition and cell polarity events in tear duct elongation. To gain a deeper understanding of the intimate relationship between BM formation and cell polarity, we generated induced pluripotent stem cells (iPSCs)-derived embryoid bodies (EBs) with a basement membrane separating the visceral endoderm (VE) and inner EB cell mass. We found that Prickle1 was highly expressed in VE of the normal EBs, and the Prickle1 mutant EBs displayed severely impaired BM. Notably, the formation of the basement membrane appeared to rely on the proper microtubule network of the VE cells, which was disrupted in the Prickle1 mutant EBs. Moreover, disruption of vesicle trafficking in the VE hindered BM secretion. Furthermore, reintroducing Prickle1 in the mutant EBs completely rescued BM formation but not the apicobasal cell polarity of the VE. Our data, in conjunction with studies by others, highlight the conserved role of Prickle1 in directing the secretion of BM components of the VE cells during embryonic germ layer differentiation, even in the absence of established general polarity machinery. Our study introduces a novel system based on iPSCs-derived EBs for investigating cellular and molecular events associated with cell polarity.

Ramipril therapy in integrin α1-null, autosomal recessive Alport mice triples lifespan: mechanistic clues from RNA-seq analysis.

The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1β1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Defining the Functional Influence of Endothelial Cell–Expressed Oncogenic Activating Mutations on Vascular Morphogenesis and Capillary Assembly

This study sought to define key molecules and signals controlling major steps in vascular morphogenesis, and how these signals regulate pericyte recruitment and pericyte-induced basement membrane deposition. The morphogenic impact of endothelial cell (EC) expression of activating mutants of Kirsten rat sarcoma virus (kRas), mitogen-activated protein kinase 1 (Mek1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), Akt serine/threonine kinase 1 (Akt1), Ras homolog enriched in brain (Rheb) Janus kinase 2 (Jak2), or signal transducer and activator of transcription 3 (Stat3) expression versus controls was evaluated, along with EC signaling events, pharmacologic inhibitor assays, and siRNA suppression experiments. Primary stimulators of EC lumen formation included kRas, Akt1, and Mek1, whereas PIK3CA and Akt1 stimulated a specialized type of cystic lumen formation. In contrast, the key drivers of EC sprouting behavior were Jak2, Stat3, Mek1, PIK3CA, and mammalian target of rapamycin (mTor). These conclusions are further supported by pharmacologic inhibitor and siRNA suppression experiments. EC expression of active Akt1, kRas, and PIK3CA led to markedly dysregulated lumen formation coupled to strongly inhibited pericyte recruitment and basement membrane deposition. For example, activated Akt1 expression in ECs excessively stimulated lumen formation, decreased EC sprouting behavior, and showed minimal pericyte recruitment with reduced mRNA expression of platelet-derived growth factor-BB, platelet-derived growth factor-DD, and endothelin-1, critical EC-derived factors known to stimulate pericyte invasion. The study identified key signals controlling fundamental steps in capillary morphogenesis and maturation and provided mechanistic details on why EC activating mutations induced a capillary deficiency state with abnormal lumens, impaired pericyte recruitment, and basement deposition: predisposing stimuli for the development of vascular malformations.

Tubular basement membrane deposits after allogeneic hematopoietic stem cell transplantation

BackgroundExtraglomerular immune complex deposition is rare and only a few membranous nephropathy cases with tubular basement membrane deposits have been reported following allogeneic hematopoietic stem cell transplantation.Case presentationWe reported a 56-year-old man with increased serum creatinine after allogeneic hematopoietic stem cell transplantation who underwent a renal biopsy. Tubular interstitial nephritis was identified on light microscope. The unique histologic features were diffuse tubular basement membrane immune complex deposition detected by both immunofluorescence and electron microscopy, while the glomerular involvement was inconspicuous. The differential diagnosis from other forms of tubular basement membrane deposition is discussed.ConclusionDiffuse granular tubular basement membrane immune complex deposition with minimal glomerular involvement is also a manifestation of renal complication in hematopoietic stem cell transplantation recipient. However, the exact mechanism and target antigen remains unknown.

Increased Matrix Metalloproteinase-1 Activation Enhances Disruption and Regression of k-RasV12–Expressing Arteriovenous Malformation-Like Vessels

This study sought to identify potential mechanisms by which k-RasV12-expressing endothelial cell (EC) tubes demonstrate an increased propensity to regress compared with controls. Activated k-Ras mutations play a role in a variety of pathological conditions, including arteriovenous malformations, which are prone to bleed, causing serious hemorrhagic complications. ECs expressing active k-RasV12 demonstrate markedly excessive lumen formation with widened and shortened tubes accompanied by reduced pericyte recruitment and basement membrane deposition, leading to deficient capillary network assembly. The current study showed that active k-Ras-expressing ECs secreted greater amounts of MMP-1 proenzyme compared with control ECs, and readily converted it to increased active MMP-1 levels through the action of plasmin or plasma kallikrein (generated from their added zymogens). Active MMP-1 degraded three-dimensional collagen matrices, leading to more rapid and extensive regression of the active k-Ras-expressing EC tubes, in conjunction with matrix contraction, compared with control ECs. Under conditions where pericytes protect control EC tubes from plasminogen- and MMP-1-dependent tube regression, this failed to occur with k-RasV12 ECs, due to reduced pericyte interactions. In summary, k-RasV12-expressing EC vessels showed an increased propensity to regress in response to serine proteinases through accentuated levels of active MMP-1, a novel pathogenic mechanism that may underlie hemorrhagic events associated with arteriovenous malformation lesions.

The Fraser Complex Proteins (Frem1, Frem2, and Fras1) Can Form Anchoring Cords in the Absence of AMACO at the Dermal–Epidermal Junction of Mouse Skin

AMACO (VWA2 protein), secreted by epithelial cells, is strongly expressed at basement membranes when budding or invagination occurs in embryos. In skin, AMACO associates with proteins of the Fraser complex, which form anchoring cords. These, during development, temporally stabilize the dermal-epidermal junction, pending the formation of collagen VII-containing anchoring fibrils. Fraser syndrome in humans results if any of the core members of the Fraser complex (Fras1, Frem1, Frem2) are mutated. Fraser syndrome is characterized by subepidermal blistering, cryptophthalmos, and syndactyly. In an attempt to determine AMACO function, we generated and characterized AMACO-deficient mice. In contrast to Fraser complex mutant mice, AMACO-deficient animals lack an obvious phenotype. The mutually interdependent basement membrane deposition of the Fraser complex proteins, and the formation of anchoring cords, are not affected. Furthermore, hair follicle development in newborn AMACO-deficient mice showed no gross aberration. Surprisingly, it appears that, while AMACO is a component of the anchoring cords, it is not essential for their formation or function.

Maturation of Nephrons by Implanting hPSC-derived Kidney Progenitors Under Kidney Capsules of Unilaterally Nephrectomized Mice.

Human pluripotent stem cell (hPSC)-derived kidney organoids may contribute to disease modeling and the generation of kidney replacement tissues. However, the realization of such applications requires the induction of hPSCs into functional mature organoids. One of the key questions for this process is whether a specific vascular system exists for nephrogenesis. Our previous study showed that short-term (2 weeks) implantation of hPSC-derived organoids below the kidney capsules of unilaterally nephrectomized and immunodeficient mice resulted in the enlargement of organoids and production of vascular cells, although signs of maturation were lacking. Organoids were induced for 15 days in vitro and then grafted below kidney capsules of the same unilaterally nephrectomized immunodeficient mouse model to examine whether medium-term (4 weeks) implantation could improve organoid maturation and vascularization, as evaluated by immunofluorescence and transmission electron microscopy. We demonstrated that after 2-4 weeks of implantation, renal organoids formed host-derived vascularization and matured without any exogenous vascular endothelial growth factor. Glomerular filtration barrier maturation was evidenced by glomerular basement membrane deposition, perforated glomerular endothelial cell development, and apical, basal podocyte polarization. A polarized monolayer epithelium and extensive brush border were also observed for tubular epithelial cells. Our results indicate that the in vivo microenvironment is important for the maturation of human kidney organoids. Stromal expansion and a reduction of nephron structures were observed following longer-term (12 weeks) implantation, suggesting effects on off-target cells during the induction process. Accordingly, induction efficiency and transplantation models should be improved in the future.

Urokinase Plasminogen Activator Deficiency Aggravates Cationic Bovine Serum Albumin–Induced Membranous Nephropathy Through T Helper Cell Type 2-Prone Immune Response in Mice

Urokinase plasminogen activator (uPA) is a crucial activator of the fibrinolytic system that modulates tissue remodeling, cancer progression, and inflammation. However, its role in membranous nephropathy (MN) remains unclear. To clarify this issue, an established BALB/c mouse model mimicking human MN induced by cationic bovine serum albumin (cBSA), with a T helper cell type 2–prone genetic background, was used. To induce MN, cBSA was injected into Plau knockout (Plau−/−) and wild-type (WT) mice. The blood and urine samples were collected to measure biochemical parameters, such as serum concentrations of immunoglobulin (Ig)G1 and IgG2a, using enzyme-linked immunoassay. The kidneys were histologically examined for the presence of glomerular polyanions, reactive oxygen species (ROS), and apoptosis, and transmission electron microscopy was used to examine subepithelial deposits. Lymphocyte subsets were determined using flow cytometry. Four weeks post–cBSA administration, Plau−/− mice exhibited a significantly higher urine protein-to-creatine ratio, hypoalbuminemia, and hypercholesterolemia than WT mice. Histologically, compared to WT mice, Plau−/− mice showed more severe glomerular basement thickening, mesangial expansion, IgG granular deposition, intensified podocyte effacement, irregular thickening of glomerular basement membrane and subepithelial deposits, and abolishment of the glycocalyx. Moreover, increased renal ROS levels and apoptosis were observed in Plau−/− mice with MN. B-lymphocyte subsets and the IgG1-to-IgG2a ratio were significantly higher in Plau−/− mice after MN induction. Thus, uPA deficiency induces a T helper cell type 2–dominant immune response, leading to increased subepithelial deposits, ROS levels, and apoptosis in the kidneys, subsequently exacerbating MN progression in mice. This study provides a novel insight into the role of uPA in MN progression.

Goodpasture syndrome and anti-glomerular basement membrane disease.

Anti-glomerular basement membrane (anti-GBM) disease is a rare life-threatening small vessel vasculitis that typically affects the capillaries of kidneys and lungs, with most of patients developing rapidly progressive crescentic glomerulonephritis, and 40%-60% concomitant alveolar haemorrhage. It is caused by the deposition in alveolar and glomerular basement membrane of circulating autoantibodies directed against antigens intrinsic to the basement membrane. The exact mechanism that induces the formation of autoantibodies is unknown, but probably environmental factors, infections or direct damage to kidneys and lungs may trigger the autoimmune response in genetically susceptible individuals. Initial therapy includes corticosteroids and cyclophosphamide to prevent autoantibodies production, and plasmapheresis to remove the circulating autoantibodies. Good renal outcomes may be achieved by a prompt treatment initiation. However, when patients present with severe renal failure requiring dialysis or with a high proportion of glomerular crescents at biopsy, renal outcomes are bad. Relapses are rare and when renal involvement is present, the suspect of concomitant diseases, such as ANCA-associated vasculitis and membranous nephropathy, should be raised. Imlifidase is showing promising results, which if confirmed will cause a paradigm shift in the treatment of this disease.

Glomerular filtration barrier modeling on a chip with tunable basement membrane deposition and 3D cultured podocytes.

In vitro investigation of a glomerular filtration barrier (GFB) remains difficult because of the inability to mimic its specialized structure, although various kidney diseases are characterized by GFB dysfunction. Here, the development of a microfluidic model that replicates the physiology of the GFB has been achieved by tunable glomerular basement membrane (gBM) deposition and 3D co-culture of podocytes with glomerular endothelial cells (gECs). By precisely controlling the thickness of the gBM, our model successfully reproduced the biphasic response of the GFB, where variations in gBM thickness influence barrier properties. Moreover, this microscale proximity of gECs and podocytes facilitated their dynamic crosstalk, which is essential for maintaining the integrity and function of the GFB. We observed that addition of gBM and podocytes enhanced barrier function of gECs by inducing up-regulation of gEC's tight junctions synergistically, and moreover, found an ultrastructure of gECs-gBM-podocytes' foot process contacting each other by confocal and TEM imaging. The dynamic interaction of gECs and podocytes played a significant role in the response to drug-induced injury and the regulation of barrier properties. Nephrotoxic injury simulated in our model helped to elucidate that the over-production of vascular endothelial growth factor A from the injured podocytes mediates GFB impairment. We believe that our GFB model can provide a valuable tool for mechanistic studies such as investigating GFB biology, comprehending disease mechanisms, and evaluating potential therapeutic approaches in a controlled and physiologically relevant environment.

New Insight on 2D In Vitro Angiogenesis Models: All That Stretches Is Not a Tube

HighlightsTube formation on MatrigelTM and tube formation in co-culture with MSCs are two different stages of angiogenesis.uPA, uPAR, Jagged1, and Notch2 are common upregulated genes for ECs on MatrigelTM, in co-culture and in dividing/migrating cells.EndMT activated at a much greater extent in ECs in a co-culture model than in a MatrigelTM assay.Only in the MatrigelTM assay are the Notch and Hippo pathway-related genes upregulated.A Matrigel-based tube formation assay is a simple and widely accepted 2D angiogenesis model in vitro. Extracellular matrix (EM) proteins and growth factors (GFs) from MatrigelTM exclusively trigger endothelial cell (EC) tubular network (ETN) formation. Co-culture of ECs with mesenchymal stromal cells (MSCs) is another and more reliable in vitro angiogenesis assay. MSCs modulate ETN formation through intercellular interactions and as a supplier of EM and GFs. The aim of the present study was to compare the expression profile of ECs in both models. We revealed upregulation of the uPA, uPAR, Jagged1, and Notch2 genes in dividing/migrating ECs and for ECs in both experimental models at 19 h. The expression of endothelial–mesenchymal transition genes largely increased in co-cultured ECs whereas Notch and Hippo signaling pathway genes were upregulated in ECs on MatrigelTM. We showed that in the co-culture model, basement membrane (BM) deposition is limited only to cell-to-cell contacts in contrast to MatrigelTM, which represents by itself fully pre-assembled BM matrix. We suggest that ETN in a co-culture model is still in a dynamic process due to immature BM whereas ECs in the MatrigelTM assay seem to be at the final stage of ETN formation.

Invasion-Associated Reorganization of Laminin 332 in Oral Squamous Cell Carcinomas: The Role of the Laminin γ2 Chain in Tumor Biology, Diagnosis, and Therapy.

Simple SummaryThe destructive growth of carcinomas is associated with crossing the border between the epithelial and the connective tissue parts of an organ. One component of this borderline, the basement membrane, is the heterotrimeric laminin 332, which mediates the adhesion of basal epithelial cells. This protein, in particular its gamma 2 chain, is fundamentally reorganized during tumor cell invasion. Specific deposition patterns of laminin 332 are also present in oral squamous cell carcinomas and have been shown to be of high diagnostic and predictive value. Furthermore, laminin 332 restructuring is associated with important tumor biological processes, e.g., stromal activation, the development of a motile phenotype, and tumor spreading. In this review, current knowledge in the field is summarized and the recommendation to consider laminin 332 as a promising grading and monitoring parameter and as a potential therapeutic target is discussed.Invasion of the connective tissue by carcinoma cells is accompanied by disintegration and reorganization of the hemidesmosomes, which connect the basement membrane to the basal epithelial cells. In terms of mediating the basement membrane, i.e., basal cell interactions, the heterotrimeric laminin 332 is the most important bridging molecule. Due to this distinct function, laminin 332, especially its gamma 2 chain, came into the focus of cancer research. Specific de novo synthesis and deposition patterns of laminin 332 are evident upon development and progression of oral squamous cell carcinomas (OSCCs). Loss from the basement membrane, cytoplasmic accumulation, and extracellular deposition are associated with crucial processes such as stromal activation and immune response, epithelial to mesenchymal transition, and tumor cell budding. In networks with components of the tumor microenvironment, altered expression of laminin 332 chains, proteolytic processing, and interaction with integrin receptors seem to promote cancer cell migration. Indeed, reorganization patterns are shown to have a high diagnostic and prognostic value. Here, we summarize the current knowledge on laminin 332 reorganization in OSCCs with special focus on its gamma 2 chain and provide, based on the current literature, evidence on its promising role as a grading and monitoring parameter and as a potential therapeutic target.

Molecular basis for pericyte-induced capillary tube network assembly and maturation.

Here we address the functional importance and role of pericytes in capillary tube network assembly, an essential process that is required for vascularized tissue development, maintenance, and health. Healthy capillaries may be directly capable of suppressing human disease. Considerable advances have occurred in our understanding of the molecular and signaling requirements controlling EC lumen and tube formation in 3D extracellular matrices. A combination of SCF, IL-3, SDF-1α, FGF-2 and insulin (“Factors”) in conjunction with integrin- and MT1-MMP-induced signaling are required for EC sprouting behavior and tube formation under serum-free defined conditions. Pericyte recruitment to the abluminal EC tube surface results in elongated and narrow tube diameters and deposition of the vascular basement membrane. In contrast, EC tubes in the absence of pericytes continue to widen and shorten over time and fail to deposit basement membranes. Pericyte invasion, recruitment and proliferation in 3D matrices requires the presence of ECs. A detailed analysis identified that EC-derived PDGF-BB, PDGF-DD, ET-1, HB-EGF, and TGFβ1 are necessary for pericyte recruitment, proliferation, and basement membrane deposition. Blockade of these individual factors causes significant pericyte inhibition, but combined blockade profoundly interferes with these events, resulting in markedly widened EC tubes without basement membranes, like when pericytes are absent.

Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin α2β1.

In Alport mice, activation of the endothelin A receptor (ETAR) in mesangial cells results in sub‐endothelial invasion of glomerular capillaries by mesangial filopodia. Filopodia deposit mesangial matrix in the glomerular basement membrane (GBM), including laminin 211 which activates NF‐κB, resulting in induction of inflammatory cytokines. Herein we show that collagen α1(III) is also deposited in the GBM. Collagen α1(III) localized to the mesangium in wild‐type mice and was found in both the mesangium and the GBM in Alport mice. We show that collagen α1(III) activates discoidin domain receptor family, member 1 (DDR1) receptors both in vitro and in vivo. To elucidate whether collagen α1(III) might cause podocyte injury, cultured murine Alport podocytes were overlaid with recombinant collagen α1(III), or not, for 24 h and RNA was analyzed by RNA sequencing (RNA‐seq). These same cells were subjected to siRNA knockdown for integrin α2 or DDR1 and the RNA was analyzed by RNA‐seq. Results were validated in vivo using RNA‐seq from RNA isolated from wild‐type and Alport mouse glomeruli. Numerous genes associated with podocyte injury were up‐ or down‐regulated in both Alport glomeruli and cultured podocytes treated with collagen α1(III), 18 of which have been associated previously with podocyte injury or glomerulonephritis. The data indicate α2β1 integrin/DDR1 co‐receptor signaling as the dominant regulatory mechanism. This may explain earlier studies where deletion of either DDR1 or α2β1 integrin in Alport mice ameliorates renal pathology. © 2022 Boys Town National Research Hospital. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

MO266: Deficiency of Urokinase-Type Plasminogen Activator Exacerbates CBSA-Induced Membranous Nephropathy in Mice

Abstract BACKGROUND AND AIMS In nondiabetic population, primary membranous nephropathy (PMN) is the most common cause of idiopathic nephrotic syndrome in the world. Urokinase-type plasminogen activator (uPA) is one of the key members of fibrinolysis system, which functions as dissolving blood clots to prevent obstruction of blood vessels. Currently, the roles of uPA in PMN remain undefined. METHOD Six-week-old male mice with Plau wild-type and knockout in BALB/c strain were used to induce MN by cationic bovine serum albumin (cBSA). Biochemistry assays of blood and urine were measured by a Fuji Dri-Chem FDC NX 500 machine. Glomerular polyanions were determined by using Colloidal Iron Stain Kit. Reactive oxygen species (ROS) was detected by immunohistochemistry stained by 4-HNE antibody. TUNEL positivity in the kidneys of mice was determined using an ApopTag® Plus Peroxidase In Situ Apoptosis Kit. The deposition of immune complex in glomeruli was examined by a transmission electron microscope. Lymphocyte subsets were determined by flow cytometry. Serum concentrations of IgG1 and IgG2a were measured by ELISA. RESULTS It has been reported that BALB/c mice is an ideal strain for mimicking human PMN. Thus, we generated uPA deficient mice (Plau-/-) in BALB/c strain by backcrossing C.129S2-Plautm1Mlg/J mice with BALB/c wild-type mice to N10. Plau-/- mice exhibited severe urinary albumin-to-creatinine ratio significantly when compared with wild-type mice at 2 weeks post-cBSA administration. Consistently, cBSA-induced Plau-/- mice displayed higher serum creatinine, hypoalbuminemia and hypercholesterolemia than wild-type mice. Histologically, Plau-/- mice presented more severed glomerular basement thickening, IgG granular deposition, intensified podocyte effacement, irregular thickening of glomerular basement membrane and subepithelial deposits than wild-type mice. Attenuated colloidal iron stain was observed in Plau-/- mice when compared with wild-type mice. Moreover, elevated ROS and apoptosis in the kidneys of Plau-/- mice were observed. B lymphocyte subsets were enriched significantly in the Plau-/- mice with MN, suggesting that pathological aggravation of MN is associated with dominant B lymphocyte population. The enriched ratio of IgG1 to IgG2a further confirmed predominant Th2 subset in Plau-/- mice. CONCLUSION uPA deficiency enhances Th2 dominant immune response, leading to enhancement of ROS and apoptosis in the kidneys, which subsequently exacerbates the progression of MN. Maintenance of uPA homeostasis may be a promising therapeutic approach for MN management.

The value of ‘raspberry bodies’ in intraoperative cytologic evaluation of adnexal masses for the diagnosis of clear cell carcinoma of the ovary: A cytological-pathological correlation

We report a case of a 48-year-old female who presented to the emergency department with pelvic/abdominal pain and a recent history of irregular periods. Pelvic ultrasound and computed tomography (CT) scan of the abdomen/pelvis revealed a 7.3 cm adnexal mass with suspicious features. During the intraoperative evaluation, a frozen section slide and a cytological smear were prepared. The cytological preparation was moderately cellular, showing cohesive groups of atypical cells with anisonucleosis, high nuclear to cytoplasmic ratio, and oval nuclei with prominent nucleoli. The tumor cells surrounded extracellular, magenta hyaline globules, forming raspberry bodies. Raspberry bodies are comprised of basement membrane deposits and are a unique finding in ovarian clear cell carcinoma. Raspberry bodies were also found in the frozen section slide, but, in comparison to the cytological preparation, were rare, difficult to identify, and resembled necrotic debris. The intraoperative diagnosis of a clear cell carcinoma is important because the surgical management will be more aggressive, as optimal tumor debulking is shown to have better overall survival. In this manuscript, we detail the intraoperative evaluation of an ovarian mass, the utility of cytological preparation and importance of identifying raspberry bodies in the evaluation of ovarian masses, and surgical management of clear cell carcinoma.