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Membranous nephropathy (MN) is an autoimmune disease caused by autoantibodies against the podocyte antigens phospholipase A 2 receptor 1 (PLA 2 R1) and thrombospondin type 1 domain containing protein 7A (THSD7A) in 80% and 2-3% of patients, respectively. THSD7A antibodies are considered to be pathogenic and highly specific for MN patients. Using an indirect immunofluorescence test (IIFT) we detected THSD7A-antibodies (titre 1:10) in the serum of a patient with high proteinuria who, however, in the kidney biopsy was diagnosed with diabetic nephropathy and MN was excluded as a possible cause of proteinuria. Different immunofluorescence assays and Western blot techniques using recombinant THSD7A (rTHSD7A) or THSD7A from different human tissues revealed that the circulating THSD7A-autoantibodies were only of the IgG3 subclass. The patient serum reacted exclusively with rTHSD7A and only when the antigen was present in reducing Western blot conditions, or on formaldehyde-fixed cells for the IIFT. Our findings show for the first time the existence of circulating THSD7A-antibodies recognizing denatured/reduced rTHSD7A, which do not react with glomerular THSD7A in vivo and are thus presumptively non-pathogenic. As a consequence, kidney biopsy or Western blot analyses of THSD7A under non-reducing conditions should be performed to confirm the diagnosis of THSD7A-associated MN, especially in cases with low THSD7A-antibody levels in the IIFT., (© 2021. The Author(s).)

Complement is an evolutionarily conserved system which is important in the defense against microorganisms and also in the elimination of modified or necrotic elements of the body. Complement is activated in a cascade type manner and activation and all steps of cascade progression are tightly controlled and regulatory interleaved with many processes of inflammatory machinery. Overshooting of the complement system due to dysregulation can result in the two prototypes of primary complement mediated renal diseases: C3 glomerulopathy and thrombotic microangiopathy. Apart from these, complement also is highly activated in many other inflammatory native kidney diseases, such as membranous nephropathy, ANCA-associated necrotizing glomerulonephritis, and IgA nephropathy. Moreover, it likely plays an important role also in the transplant setting, such as in antibody-mediated rejection or in hematopoietic stem cell transplant associated thrombotic microangiopathy. In this review, these glomerular disorders are discussed with regard to the role of complement in their pathogenesis. The consequential, respective clinical trials for complement inhibitory therapy strategies for these diseases are described., (© 2021. The Author(s).)

Proteases play a central role in regulating renal pathophysiology and are increasingly evaluated as actionable drug targets. Here, we review the role of proteolytic systems in inflammatory kidney disease. Inflammatory kidney diseases are associated with broad dysregulations of extracellular and intracellular proteolysis. As an example of a proteolytic system, the complement system plays a significant role in glomerular inflammatory kidney disease and is currently under clinical investigation. Based on two glomerular kidney diseases, lupus nephritis, and membranous nephropathy, we portrait two proteolytic pathomechanisms and the role of the complement system. We discuss how profiling proteolytic activity in patient samples could be used to stratify patients for more targeted interventions in inflammatory kidney diseases. We also describe novel comprehensive, quantitative tools to investigate the entirety of proteolytic processes in a tissue sample. Emphasis is placed on mass spectrometric approaches that enable the comprehensive analysis of the complement system, as well as protease activities and regulation in general., (© 2021. The Author(s).)

Bevacizumab is a humanized monoclonal IgG1 antibody, which neutralizes vascular endothelial growth factor and is used for treating multiple cancer types. As a known and frequent adverse event, this therapy can lead to renal damage including proteinuria and nephrotic syndrome. In a retrospective approach, we analyzed 17 renal biopsies from patients receiving bevacizumab treatment. We observed a distinctive histopathological pseudothrombotic pattern different from the previously reported thrombotic microangiopathy. Since this pattern includes some features similar to acute and chronic thrombotic microangiopathy, focal segmental glomerulosclerosis and cryoglobulinemic membranoproliferative glomerulonephritis, biopsies with these diagnoses were included for comparison. Clinical, laboratory, light microscopic, immunohistochemical (including a proximity ligation assay), proteomic and electron microscopic features were assessed. Nephrotic syndrome was present in 15 of the 17 bevacizumab-treated patients. All 17 displayed a patchy pattern of variably PAS-positive hyaline pseudothrombi occluding markedly dilated glomerular capillaries in their biopsies. Mass spectrometry-based proteome analysis revealed a special protein pattern demonstrating some features of thrombotic microangiopathy and some of cryoglobulinemic glomerulonephritis, including a strong accumulation of IgG in the pseudothrombi. Proximity ligation assay did not show interaction of IgG with C1q, arguing for accumulation without classic pathway complement activation. In contrast to thrombi in thrombotic microangiopathy cases, the hyaline pseudothrombi did not contain clusters of CD61-positive platelets. Electron microscopy of bevacizumab cases did not show fibrin polymers or extensive loss of podocyte foot processes. Even though cases of bevacizumab-associated microangiopathy share some features with thrombotic microangiopathy, its overall histopathological pattern is quite different from acute or chronic thrombotic microangiopathy cases. We conclude that bevacizumab therapy can lead to a unique hyaline occlusive glomerular microangiopathy, likely arising from endothelial leakage followed by subendothelial accumulation of serum proteins. It can be diagnosed by light microscopy and is an important differential diagnosis in cancer patients with nephrotic syndrome.

C3 glomerulopathy describes a spectrum of disorders with glomerular pathology associated with C3 cleavage product deposition and with defective complement action and regulation (Fakhouri et al., 2010; Sethi et al., 2012b). Kidney biopsies from these patients show glomerular accumulation or deposition of C3 cleavage fragments, but no or minor deposition of immunoglobulins (Appel et al., 2005; D'Agati and Bomback, 2012; Servais et al., 2007; Sethi and Fervenza, 2011). At present the current situation asks for a better definition of the underlining disease mechanisms, for precise biomarkers, and for a treatment for this disease. The complement system is a self activating and propelling enzymatic cascade type system in which inactive, soluble plasma components are activated spontaneously and lead into an amplification loop (Zipfel and Skerka, 2009). Activation of the alternative pathway is spontaneous, occurs by default, and cascade progression leads to amplification by complement activators. The system however is self-controlled by multiple regulators and inhibitors, like Factor H that control cascade progression in fluid phase and on surfaces. The activated complement system generates a series of potent effector components and activation products, which damage foreign-, as well as modified self cells, recruit innate immune cells to the site of action, coordinate inflammation and the response of the adaptive immune system in form of B cells and T lymphocytes (Kohl, 2006; Medzhitov and Janeway, 2002; Ogden and Elkon, 2006; Carroll, 2004; Kemper and Atkinson, 2007; Morgan, 1999; Muller-Eberhard, 1986; Ricklin et al., 2010). Complement controls homeostasis and multiple reactions in the vertebrate organism including defense against microbial infections (Diaz-Guillen et al., 1999; Mastellos and Lambris, 2002; Nordahl et al., 2004; Ricklin et al., 2010). In consequence defective control of the spontaneous self amplifying cascade or regulation is associated with numerous human disorders (Ricklin and Lambris, 2007; Skerka and Zipfel, 2008; Zipfel et al., 2006). Understanding the exact action and regulation of this sophisticated homeotic cascade system is relevant to understand disease pathology of various complement associated human disorders. Furthermore this knowledge is relevant for a better diagnosis and appropriate therapy. At present diagnosis of C3 glomerulopathy is primarily based on the kidney biopsy, and histological, immmunohistological and electron microscopical evaluation (D'Agati and Bomback, 2012; Fakhouri et al., 2010; Medjeral-Thomas et al., 2014a,b; Sethi et al., 2012b). The challenge is to define the actual cause of the diverse glomerular changes or damages, to define how C3 deposition results in the reported glomerular changes, the location of the cell damage and the formation of deposits., (Copyright © 2015. Published by Elsevier Ltd.)

Background: Renal amyloidosis results from protein misfolding and leads to progressive renal insufficiency. Few data are available concerning the relevance of the histomorphological patterns and the dynamics of the disease process., Methods: Cases of renal amyloidosis in native kidney biopsies (n = 203) were retrospectively evaluated for the pattern of amyloid distribution, the extent of glomerular amyloid deposition and the amount of interstitial fibrosis and tubular atrophy. One hundred and fifty-eight cases were characterized by immunohistochemistry to determine the biochemical amyloid type. Morphological findings were correlated with available clinical data., Results: According to the predominant site of amyloid deposition, 84.6% showed a glomerular, 9.4% a vascular and 6% a tubulointerstitial distribution pattern. Within the glomeruli, amyloid was initially deposited in a focal segmental fashion that became diffuse and global in later stages. Most cases were identified as AL lambda (84/158) or AA (68/158). There was no correlation between the biochemical type and the distribution pattern. Serum creatinine correlated well with interstitial fibrosis and tubular atrophy and proteinuria with the glomerular amyloid load., Conclusions: The relevance of the different distribution patterns is unclear at the moment, but they may be due to the physicochemical properties of the amyloid fibrils in a given patient. This may become important in future anti-fibrillar therapies.

Injury and loss of podocytes are leading factors of glomerular disease and renal failure. The postmitotic podocyte is the primary glomerular target for toxic, immune, metabolic, and oxidant stress, but little is known about how this cell type copes with stress. Recently, autophagy has been identified as a major pathway that delivers damaged proteins and organelles to lysosomes in order to maintain cellular homeostasis. Here we report that podocytes exhibit an unusually high level of constitutive autophagy. Podocyte-specific deletion of autophagy-related 5 (Atg5) led to a glomerulopathy in aging mice that was accompanied by an accumulation of oxidized and ubiquitinated proteins, ER stress, and proteinuria. These changes resulted ultimately in podocyte loss and late-onset glomerulosclerosis. Analysis of pathophysiological conditions indicated that autophagy was substantially increased in glomeruli from mice with induced proteinuria and in glomeruli from patients with acquired proteinuric diseases. Further, mice lacking Atg5 in podocytes exhibited strongly increased susceptibility to models of glomerular disease. These findings highlight the importance of induced autophagy as a key homeostatic mechanism to maintain podocyte integrity. We postulate that constitutive and induced autophagy is a major protective mechanism against podocyte aging and glomerular injury, representing a putative target to ameliorate human glomerular disease and aging-related loss of renal function.

Injury of the glomerular filter causes proteinuria by disrupting the sensitive interplay of the glomerular protein network. To date, studies of the expression and trafficking of glomerular proteins have been mostly limited to in vitro or histologic studies. Here, we report a novel in vivo biotinylation assay that allows the quantification of surface expression of glomerular proteins in mice. Kidneys were perfused in situ with biotin before harvest. Afterwards glomeruli were isolated and lyzed. The protein of interest was separated by immunoprecipitation and the amount of surface-expressed protein was quantified by Western blot analysis with streptavidin staining. As proof-of-concept, we examined the presence of nephrin in the slit diaphragm in two well-established murine models of proteinuric kidney disease: nephrotoxic nephritis and adriamycin nephropathy. In proteinuric animals, significantly less nephrin was detected in the slit diaphragm. When proteinuria decreased once again during the course of disease, the amount of surface nephrin returned to the baseline. Our present results suggest that our assay is a valuable tool to study the glomerular filter in proteinuric kidney diseases. Note that the assay is not limited to proteins expressed in the slit diaphragm, and all surface proteins that are accessible to biotin perfusion and immunoprecipitation qualify for this analysis. [ABSTRACT FROM AUTHOR]

Background: Interdigitating podocyte foot processes are an essential part of the kidney filtration barrier. They form highly specialized cell-cell contacts that are ultrastructurally described as a slit diaphragm (SD). The SD is composed of a multiprotein complex that contains features of adherens as well as tight junctions which link the large extracellular domain of nephrin to the cytoskeleton. One tight junction-associated protein that has been shown to be localized within the SD complex is MAGI2 (membrane-associated guanylate kinase inverted 2), which is specifically expressed in podocytes. The aim of this work was to study the expression of MAGI2 in glomerular diseases and to evaluate whether a diff erent regulation of this protein occurs in diff erent glomerulopathies. Additionally, we wanted to investigate the infl uence of MAGI2 and nephrin on their relative localization. Methods: We investigated the Magi2 regulation in three different rodent models of glomerulopathy as well as in the genetic focal segmental glomerulosclerosis (FSGS)-like zebrafish model. MAGI2 expression was evaluated in kidney biopsies of patients affected by minimal change disease (MCD), primary and secondary focal segmental glomerulosclerosis in comparison with healthy controls. Immunostaining was performed and slides were imaged using confocal laser scanning and super-resolution 3D-structured illumination microscopy (3D-SIM). Filtration slit density (FSD) was determined on human biopsies by PEMP (podocyte exact morphology measurement procedure) [1] and MAGI2/Nephrin ratio measurements were performed with a customized macro for the ImageJ-based platform FIJI. Using LC-MS/MS, RNA sequencing, and Taqman RT-qPCR, expression studies were performed in murine glomeruli freshly isolated or in vitro de-differentiated over time (GlomAssay) [2]. To evaluate the coregulation of Magi2 and nephrin in the zebrafish model, the respective orthologues (nphs1 and magi2a) were knocked-out using CRISPR/Cas9. Transmission electron microscopy was used to investigate the effects of the knockout from an ultrastructural point of view. The expression of these two proteins in both strains was analyzed by immunofl uorescence staining and proteinuria was assessed using an automated screening approach. Results: MAGI2 was expressed in a linear pattern along the filtration slit and colocalized with nephrin in both healthy filtration slit and eff aced areas regardless of the species (human, mouse, rat and zebrafi sh). Magi2 was uniformly down-regulated in animal models of glomerulopathy as well as in de-differentiated podocytes. A reduction of MAGI2 was found in human biopsies of patients suffering from primary FSGS, but not in MCD and secondary FSGS. Transmission electron microscopy revealed podocyte foot process effacement in both CRISPR/Cas9 zebrafi sh knockout models, while the morphology of the glomerular basement membrane and glomerular endothelial cells was well preserved. Additionally, Nphs1 expression was significantly reduced in magi2a-KO larvae, whereas interestingly Magi2a expression was unchanged in nphs1-KO larvae. Conclusions: Here, we have demonstrated that the expression of MAGI2 in the glomerulus is associated with the expression of nephrin in all the species investigated. By using knockout larvae, we showed that Nphs1 expression is dependent on Magi2a and not vice versa. MAGI2 is downregulated in glomerular diseases, presumably as a consequence of podocyte dedifferentiation. Finally, the expression of MAGI2 seems to be specifically regulated depending on the podocytopathy investigated, suggesting a role for this protein to distinguish between different glomerulopathies. [ABSTRACT FROM AUTHOR]