Atheroembolic Kidney Disease: The Under-recognized Silent Killer

Role of proteases in inflammation 

Enzymatic proteolysis controls myriad physiological and pathophysiological processes, such as differentiation (Canalis et al. 2003), development (Kopan and Ilagan 2009), apoptosis (Taylor et al. 2008), hormone activation (Hampton 2002), neurodegeneration (O’Brien and Wong 2011), and cancer (Kessenbrock et al. 2010). Protease activity is essential for the propagation and resolution of coagulation and inflammation. In inflammation, rapid protease activity is a key component of the innate immune system and contributor to the microenvironment, and responsible for tissue remodeling. Several proteases are active within the inflammatory microenvironment, such as cathepsins (Joyce and Pollard 2009), urokinase PAR receptors (Andreasen et al. 1997; Joyce and Pollard 2009), matrix metalloproteinases (MMPs) (Prudova and Overall 2010; auf dem Keller et al. 2013; Eckhard et al. 2016), lysozyme (Satoskar et al. 2020), and the complement system (Ricklin et al. 2010). Recently, it has been shown that many of the proteases also target not only their direct substrates but also display unexpected substrates, modifying additional protein factors, which in turn interact with one another in a proteolysis-dependent manner. This hypothesis of a tightly regulated and fate-determining “protease web” (Fortelny et al. 2014; Rinschen et al. 2018b) postulates that proteases form functional networks with many interactions to govern pathophysiological processes. This notion expands the traditional and widely accepted concept of unidirectional proteolytic cascades, such as the initiation of apoptosis by caspase-8/-9-mediated proteolytic activation of caspase 3 (Porter and Jänicke 1999). With 588 and 628 proteases.

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The complement system is a key serum protease systemThe complement system is a well-studied and relevant proteolytic system whose activation is widely accepted to be triggered during glomerular kidney disease. The complement system is an essential part of the innate immune system and is vital for maintaining tissue homeostasis (Ricklin et al. 2010; Bajic et al. 2015). It can identify and opsonize targets, including invading microbes, immune complexes, necrotic tissue, and apoptotic cells, and hereafter facilitate their safe removal via phagocytosis (Merle et al. 2015b). The proteolytic cascades of the complement system are tightly regulated (Fig. 1) by several proteins (Merle et al. 2015a; Schmidt et al. 2016). If the delicate balance between activation and regulation is tipped, the system may act as a double-edged sword causing self-damage manifesting as various immune-mediated and inflammatory diseases (Bajic et al. 2015). Initiation of the complement system may occur through three pathways (Fig. 1), termed the classical pathway (CP), lectin pathway (LP), and alternative pathway (AP). While the CP and LP have specific initiating molecules (antibodies bound to antigens and patterns of carbohydrate structures, respectively), the AP is triggered by the spontaneous activation of complement factor C3 in the fluid phase. The pathways converge at the cleavage of complement factor C3 into C3b and C3a, resulting in (1) opsonization of pathogens by split products of C3, (2) cell lysis via formation of the membrane attack complex, and (3) inflammation by recruitment of inflammatory cells such as neutrophils by pro-inflammatory mediators like C5a (Merle et al. 2015a). While it is widely acknowledged that the complement system plays an integral role in disease progression and can guide clinical diagnosis and classification, there is a lack of understanding of which complement proteins or functional protein fragments, also termed proteoforms (van der Burgt and Cobbaert 2018), are best suited as sensitive and specific diagnostic biomarkers when measured as part of routine clinical care in inflammatory kidney disease. The complexity of the complement system, which encompasses approximately 50 proteins in circulation, demands a holistic and quantitative approach to identify the most important contributors and markers of inflammation (Ricklin et al. 2010). For an overview of appropriate measurement strategies, we refer to other reviews (Ekdahl et al. 2018). The most commonly used assays are nephelometry and turbidimetry which utilize polyclonal antibodies against a specific analyte (e.g., C3 or C4). Notably, comprehensive approaches regarding the high-throughput profiling of clinical samples are currently missing. Clinical trials with complement inhibitors such as CCX168 targeting C5aR (clinical trial code NCT02994927), OMS721 targeting MASP2 (NCT03608033), or C1INH targeting C1r and C1s (NCT02547220) are currently under investigation and may potentially be integrated as new treatments of selected diseases. Currently, 28 clinical trials, including six phase III trials, all with relevance to glomerular kidney disease, are ongoing and the C5 inhibitor Eculizumab is already available on the market. Cistanche has the function of tonifying the kidney.

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The complement system in membranous nephropathy (MN) is antibody-mediated proteinuric kidney disease. Glomerular complement deposition can be readily detected by immunofluorescenceand mass spectrometry-based approaches in patient biopsies (Person et al. 2019; Ravindran et al. 2020). The proposed pathophysiological mechanism of MN derives from investigations in a rat model of the disease, the so-called passive Heymann nephritis (PHN) (Heymann 1952). In this model, podocyte-directed heterologous antibodies from sheep (or other species) are transferred to rats, causing the formation of subepithelial immune deposits, which are considered the morphological hallmark sign of MN and proteinuria. The injected antibodies induce the local activation of the complement system with the formation of the membrane attack complex C5b-9 (Kerjaschki 1992). In PHN, blocking the complement system by means of cobra venom factor was reported to completely prevent proteinuria development (Salant et al. 1980). However, other experimental reports described the development of MN in the absence of complement deposition (Tomas et al. 2016, 2017) and after pharmacological complement depletion (Leenaerts et al. 1995), challenging the concept of the complement system as the sole mediator of cell injury and proteinuria in MN. In patients with MN, autoantibodies against two podocyte antigens have been identified, the phospholipase A2 receptor 1 (PLA2R1) and thrombospondin type-1 domain-containing 7A (THSD7A) (Beck et al. 2009; Tomas et al. 2014). The classical pathway of the complement system is activated by binding an antibody to an antigen. This mechanism can in principle apply to an antibody-mediated disease such as MN. However, anti-PLA2R1 and anti-THSD7A autoantibodies are dominant in the IgG4 subclass, which is the IgG subclass with the least C1q binding capacity (Vidarsson et al. 2014), indicating that the alternative and lectin pathways may play a role in the pathogenesis of MN (Seikrit et al. 2018; Zhang et al. 2020). However, patients with PLA2R1- and THSD7A-associated MN usually have autoantibodies of C1q-binding non-IgG4 subclasses as well, principally enabling the activation of the complement system via the classical pathway (Huang et al. 2013; von Haxthausen et al. 2018). A study published while this paper was in the review showed that IgG4 glycosylation in PLA2R1- associated MN may be responsible for the activation of the lectin pathway, and subsequent activation of podocyte proteolytic pathways via cathepsin proteases (Haddad et al. 2020). Taken together, the presence of complement components at the site of tissue injury is undoubted in MN, but whether this contributes to MN pathogenesis or simply represents an epiphenomenon is still unclear today. Novel methodological approaches are needed to clarify the role of complement in MN.

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Conclusion Proteases are key modulators of glomerular function, and the complement is an important proteolytic system that communicates between the epithelia and the innate immune system. While several inflammatory kidney diseases show that proteolysis is active and can be targeted genetically, it remains under investigation—both clinically and preclinically—if protease inhibition can emerge as a therapeutic strategy in glomerular inflammatory kidney disease. Within this context, novel aspects of complement system characterization can be useful. Traditionally regarded as a simple proteolytic cascade, the complement system exhibits increasingly recognized complex interactions with other proteolytic enzymes and inhibitors (auf dem Keller et al. 2013), resulting in severe challenges for the development of reliable parameters for complement-based diagnosis and patient stratification in kidney disease. Part of the current limitations is analytical in nature, given the fact that more than 50 proteins, each with multiple proteoforms with distinct functions and widely different abundance, make up the complement system. No consensus has been reached on what to measure when to measure, and how to measure complement activation (Ekdahl et al. 2018), and the non-canonical effects of complement proteases have not yet been systematically analyzed in kidney disease. Further proteolytic systems, such as coagulation and fibrinolysis, on the other hand, are not commonly investigated despite possible interactions (Amara et al. 2010; Oikonomopoulou et al. 2012). Therefore, further improvement of mass spectrometry-based and chemical biology techniques are needed to further and deeper profleproteases’ action in inflammatory kidney disease. Analysis of the proteolytic microenvironment in glomerular disease, including the complement system, may help stratify patients for therapeutic intervention, for instance, by complement inhibitors. Candidates for deep proteolytic analysis proteomics include membranous nephropathy and lupus nephritis. Here, integrated proteomics profiling of human kidney biopsies and serum samples will lead to an increased understanding of the pathobiology of protease-driven inflammation and might be used to stratify and prioritize patients for therapy with complement inhibition. Cistanche has the function of enhancing kidney function.

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