New insights into the immune functions of podocytes: the role of complement

Podocytes are highly specialized epithelial cells of the glomerulus and represent a major component of the GFB [1]. They have a complex architecture including a large cell body facing the urinary space and an interdigitating network of extensions (primary and secondary processes) terminating as (tertiary) foot processes on the GBM [2].

Normal podocyte function is guaranteed by a sophisticated actin cytoskeleton, mainly localized within the foot processes [3]. Podocytes are characterized by a highly complex architecture regulated by multiple proteins, grouped into two main podocyte structures: the slit diaphragm (SD) and focal adhesions (FA). The SD is a unique highly specialized cell–cell junction between two podocyte foot processes (Fig. 1), including key proteins like nephrin, podocin, or synaptopodin [4, 5]. The SD represents not only a size-selective barrier to prevent filtration of large macromolecules but also a signalling platform with critical functions, such as regulation of the actin cytoskeleton and initiation of signalling pathways to modulate the plasticity of foot processes [6]. FA are complex structures which are able to connect the actin cytoskeleton of foot processes to the GBM, thanks to two main molecular components: integrins and GTPases.

Besides contributing to the GFB, podocytes play important functions such as synthesis and repair of the GBM (together with endothelial cells), production of VEGF, and platelet-derived growth factor (PDGF) [6‐9]. Moreover, growing literature suggests that podocytes have many functions of the innate and adaptive immune systems [10‐13]. They express cytokine and chemokine receptors to respond to a variety of soluble mediators. They are also able to synthesize inflammatory mediators, such as interleukin-1 (IL-1), which may contribute to local inflammation. Evidence in literature suggests a possible role in the adaptive immune system too, as antigen-presenting cells (APC) to initiate specific T-cell responses, like dendritic cells or macrophages [14, 15].

Furthermore, podocytes express several complement components, such as complement receptor type 1 (CR1) and type 2 (CR2) and complement regulators like CD46, CD55, or CD59, and they can produce complement proteins locally, including complement component 3 (C3) and CFH [16‐18]. Nevertheless, the role of complement components expressed or secreted by podocytes in regulation of the local complement reaction is not fully understood.

Podocyte injury is involved in the pathophysiology of several glomerular diseases, like immune-complex glomerulonephritis, minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), and collapsing glomerulopathy [19, 20], and evidence from the literature suggests that the complement system could be primary or secondary involved in the podocyte damage [21‐23].

Increasing evidence suggests that podocytes play a role in the innate immune response because of their expression of Toll-like receptors (TLRs), especially TLR4, a subtype able to recognize bacterial lipopolysaccharide (LPS). Those receptors are upregulated in animal models of cryoglobulinemic membranoproliferative glomerulonephritis, and they could mediate glomerular damage by modulating expression of chemokines [12].

TLRs are located on the cell surface or intracellularly and can be expressed by different types of cells, such as dendritic cells, macrophages and monocytes, fibroblasts, B and T cells, and endothelial and epithelial cells. They play an essential role by recognizing pathogen-associated molecular patterns; in particular, cell surface TLRs can mainly recognize microbial membrane components such as LPS, lipids, and proteins, while intracellular TLRs mainly recognize nucleic acids from bacteria and viruses [24]. In addition, TLRs can be activated by endogenous ligands released during stress or tissue injury, such as heat shock proteins, mRNA, and necrotic debris [25]. Cultured human podocytes constitutively express cell surface TLRs (i.e., TLR1, 2, 3, 4, 5, 6, and 10) [26], suggesting a possible role in the defense against microbial agents; however, de novo expression of intracellular TLRs subtype has also been reported in podocytes of patients with glomerular disease. In particular, puromycin aminonucleoside (PAN), commonly used to induce a nonimmune podocyte injury in vitro, can upregulate TLR9 intracellular expression and activate NF-κB and p38 MAPK in human immortalized podocytes, utilizing endogenous mtDNA as TLR9 ligand to facilitate podocyte apoptosis [27]. This would suggest a bivalent role of TLRs in podocytes, both as major players in response to foreign pathogens and mediators of podocyte damage.

Moreover, podocytes can express MHC class I and II genes [28, 29], as well as B7-1 (or CD80, involved in T-cell activation) [15, 30] and FcRn (IgG and albumin transport receptor, used by podocytes to internalize IgG from the GBM) [31, 32]. In particular, MHC class II expression on podocytes is required for the development of immune-mediated renal injury, as MHC II presentation by podocytes is necessary to induce the CD4 + T-cell-driven glomerular disease [14]. It is reported that these cells can act as antigen-presenting cells (APC), as they can express several macrophagic-associated markers [33, 34], and they are able to process antigens to initiate specific T-cell responses [15], supporting their multifunctional role in the immunological pathogenesis of glomerular diseases.

Furthermore, expression of functional chemokine receptors (CCR4, CCR8, CCR9, CCR10, CXCR1, CXCR3, CXCR4, and CXCR5) has been demonstrated in cultured human podocytes [35, 36]. Chemokines are small chemoattractant cytokines released by innate immune cells (i.e., neutrophils, eosinophils, macrophages, dendritic cells, natural killer cells), as well as endothelial and epithelial cells. They play a central role in inflammation and immune cell recruitment by guiding circulating leukocytes to inflammation or damage site [37, 38]. They also promote cell growth and tumor angiogenesis and are able to modulate apoptosis by binding G-protein-coupled receptors (GPCRs) on the surface of immune cells. Chemokine receptors are expressed in leukocytes, as well as non-hemopoietic cells, such as endothelial and epithelial cells [39].

CXCR1, CXCR3, and CXCR5 chemokine receptors have been identified in podocytes from kidney biopsies of patients with primary membranous nephropathy (PMN), while they were not expressed in healthy kidneys. Huber et al. suggested that podocyte CXCRs activation may contribute to GFB disruption and onset of proteinuria in PMN through hyperactivation of NADPH oxidases and oxygen radicals production [36].

Podocytes are involved in the inflammatory response of several human glomerulopathies, as suggested by their ability to produce pro-inflammatory cytokines like IL-1α and IL-1β [40, 41]. It has been reported that they can express inflammasome components, like NOD-like receptor (NLR) family proteins, which contribute to inflammatory response in the local kidney in primary glomerular diseases like lupus nephritis (LN) [42].

Podocytes are also known to secrete and/or express several complement proteins and regulators, suggesting local activation of the complement cascade. Expression of complement genes, including C1q, C1r, C2, C3, C3a receptor (C3aR), C5a receptor (C5aR), C7, CR1, and CR2, has been detected in cultured podocytes under normal physiological conditions, with increased local synthesis of complement proteins following podocyte injury [16, 17]. On the other side, complement regulators have been identified too, both membrane-bound (CD46, CD55, CD59) and soluble (CFI and CFH) forms. In particular, podocytes can express CFH locally to clear subendothelial immune complex deposits [43]. The fact that podocytes are able to produce complement components, including regulators, might have a relevant impact on podocytopathies where the complement system plays a pathogenic role. The balance between local complement activation and regulation is important to maintain the glomerular environment, as podocytes could become both target and source of injury, contributing to local complement activation and amplifying their own damage [44, 45].

A summary of the main immune functions of podocytes are summarized in Table 1.

The complement system, classically described as part of the innate immune system, represents indeed a functional bridge between innate and adaptive immunity. It consists of more than 30 plasma or membrane-anchored proteins and regulators which play a role in inflammation, opsonization and lysis of pathogens, clearance of apoptotic cells, and enhancement of both innate and adaptive immunity [46‐48]. It can be activated by three different pathways, the classical, the lectin, and the alternative pathway [49, 50], which are tightly regulated by several complement components, like the membrane-bound proteins CD46, CD55, and CD59 and the soluble CFH, to prevent uncontrolled complement hyperactivation [51]. All three pathways induce a proteolytic cascade leading to a shared terminal pathway with subsequent membrane attack complex (MAC) assembly in the cell plasma membrane. Once inserted in the lipid bilayer, MAC forms a stable pore with ~ 10 nm diameter generating several intracellular signals, which have been characterized by both in vivo and in vitro models as summarized in Table 2 [52].

Podocytes play a critical role to ensure the glomerular homeostasis. Over the years, growing literature highlighted the multiple and complex biological functions of these pericytes-like epithelial cells, which are much more than a supporting component of the GFB [1, 80‐82].

Several authors described them as “immune podocytes,” to underline their properties as both innate and adaptive immune cells [10, 13, 15]. Understanding their complex biology is essential to unravel the pathogenic mechanisms of several glomerular diseases, where podocyte injury represents a common denominator.

The role of the complement system in podocyte injury has also been evaluated in a multitude of kidney disorders, such as membranous nephropathy, lupus nephritis, HUS, FSGS, and several more [45, 83‐90]. The effects of complement activation on podocytes can vary based on the disease pathophysiology, as well as based on the initial trigger, which could induce lytic versus sub-lytic effects. Interestingly, podocytes have developed several protective mechanisms to escape the complement attack, such as autophagy, internalization mechanisms like endocytosis, and expression of complement regulators, and the balance between injury and defense mechanisms can ultimately determine the destiny of the podocyte cell [65, 69, 91].

Future studies, both in vitro and in vivo, are needed to better understand the role of complement activation in podocytopathies and the rationale for the use of anti-complement therapies in conditions where the complement system appears as main driver of the disease.