Background

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.

Fig. 1
figure 1

Main components of the slit diaphragm and podocyte-endothelial cell cross talk in healthy versus damaged podocytes. Podocyte slit diaphragm, glomerular basement membrane (GBM), and endothelial cells are the main components of the glomerular filtration barrier. Podocyte effacement/detachment, secondary to mechanical, oxidative, and/or immunologic triggers, is characterized by loss of silt diaphragm integrity, disruption of actin cytoskeleton and focal adhesions, and interruption of the physiological podocyte-endothelial cell cross talk (dashed arrows). Abbreviations: GBM, glomerular basement membrane; Ang, angiopoietin; ANGPTL, angiopoietin-like protein; IGF, insulin-like growth factor; IGFBP-rP1, insulin-like growth factor-binding protein-related protein 1; ET-1, endothelin-1; HGF, hepatocyte growth factor; IL-1, interleukin-1; NO, nitric oxide; TNF-a, tumor necrosis factor-a; VEGF, vascular endothelial growth factor

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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) [6,7,8,9]. Moreover, growing literature suggests that podocytes have many functions of the innate and adaptive immune systems [10,11,12,13]. 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 [16,17,18]. 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 [21,22,23].

The immune podocyte: innate and adaptive functions

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.

Table 1 Summary of (potential and recognized) podocyte immune functions
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Podocyte and complement system

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 [46,47,48]. 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].

Table 2 Signalling pathways activated by MAC (adapted from Takano et al. (2013). Seminars in Nephrology. Reference [52]
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Sublytic effects of complement activation on podocytes

Mechanical, oxidative, and immunologic stress can cause podocyte damage and subsequently affect the integrity of glomerular barrier. Complement activation with sublytic MAC formation on podocytes is an example of immunologic stress, which can trigger downstream pathways including protein kinases, lipid metabolism, cytokine production, ROS generation, growth factor signal transduction, endoplasmic reticulum stress, and the ubiquitin–proteasome system, eventually leading to disruption of the podocyte actin cytoskeleton and subsequent cell detachment [53].

More in details, evidence suggests that sublytic amount of MAC on the podocyte surface can induce calcium influx through the membrane pore, as well as calcium release from the intracellular storages, eventually leading to increased intracellular calcium which can activate multiple pathways, such as protein kinase signalling, and in particular protein kinases C (PKC) responsible for membrane vesiculation and internalization of MAC channels [52, 54,55,56,57], as suggested by reduction of MAC endocytosis by inhibiting PKC pathway [58].

It is well known that Ca2+ signalling in healthy podocytes is mainly mediated by angiotensin II and TRPC5 and 6 (nonselective cationic channels, downstream of angiotensin II signalling) [59]; interestingly, TRPC6 can play a dual role, as it has been shown that acute activation of this channel is able to protect podocytes from complement-mediated injury, while gain-of-function mutations/chronic hyperactivation can affect the SD and/or foot processes morphology leading to glomerular diseases, such as FSGS [60].

It has also been described that sublytic MAC can induce transactivation of receptor tyrosine kinases at the plasma membrane of cultured podocytes, resulting in activation of the Ras-extracellular signal-regulated kinase (ERK) pathway and phospholipase C-γ1. Transactivated receptor tyrosine kinases could play as scaffold for proteins assembly and/or activation, inducing activation of downstream pathways, either dependently or independently the increased cytosolic calcium levels [54, 61].

Other pathways activated by MAC formation on podocyte surface involve arachidonic acid (AA) release by cytosolic phospholipase A2-α (cPLA2), inducing AA metabolism to prostanoids, as described by Cybulsky et al. [62]. Eicosanoids can play a role in complement-mediated podocyte injury, as supported by experimental models of membranous nephropathy. Despite the exact mechanisms of glomerular damage are still unclear, cytotoxic consequences of cPLA2 activation could include release of free fatty acids and lysophospholipids, as well as ions influx, which could ultimately affect the energy machinery [63].

ROS production has also been described in podocytes exposed to sublytic amounts of MAC; both cultured and in vivo podocytes express components of the NADPH oxidase, a complex enzyme able to reduce molecular oxygen to the superoxide anion, which is further metabolized to other ROS [52]. Lipid peroxidation and changes in the podocyte membrane composition, as well as in the GBM components, have been reported as consequence of ROS production. Moreover, inhibition of ROS and/or lipid peroxidation resulted in reduced proteinuria in animal models of membranous nephropathy, suggesting their pathogenic role in glomerular damage [64].

Endoplasmic reticulum (ER) stress with accumulation of misfolded proteins and subsequent increase of the ubiquitin–proteasome system has been reported as additional response to complement-mediated injury, as possible protective response of podocytes to ongoing complement attack [65].

Sublytic MAC deposition on podocytes can also induce DNA damage, both in vitro and in vivo models, as demonstrated by Pippin et al. [66]. The authors also described that sublytic MAC-induced podocyte injury was associated with an increase in specific cell cycle-related genes, including p53, p21, growth-arrest DNA damage-45, and checkpoint kinase-1 and 2, leading to cell cycle arrest and podocyte growth suppression. This could explain why podocyte proliferation is limited following immune-mediated injury.

Consequences of complement activation on podocyte energy metabolism

The effects of complement activation on podocyte energy machinery are not fully understood. Brinkkoetter et al. demonstrated that podocyte metabolism is somewhat different from other type of cells, as it primarily relies on anaerobic glycolysis and the transformation of glucose to lactate [67]. More in details, the authors showed a significantly lower mitochondrial density per cell area, compared to other type of renal cells (i.e., renal tubular cells). Also, glomeruli stained for mitochondrial enzyme superoxide dismutase 2 (SOD2) and the glycolytic enzyme pyruvate kinase M2 (PKM2) confirmed the perinuclear localization of mitochondria (and their almost complete absence in secondary and tertiary processes), while PKM2 was ubiquitous, suggesting podocyte processes as a large compartment of anaerobic glycolysis. They also used Tfam (mitochondrial transcription factor A) knockout mice to demonstrate that loss of mitochondrial transcription and lack of the oxidative phosphorylation machinery do not induce podocyte disease. In addition, transient knockdown of Tfam in human podocytes significantly reduced mitochondrial respiration, while anaerobic glycolysis was significantly increased allowing a normal podocyte function.

It has been demonstrated that sublytic complement-mediated injury induces reduction of intracellular ATP, in addition to reversible disruption of actin stress fibers and focal adhesions, mainly due to dephosphorylation (instead of degradation) of focal contact proteins, as described by Topham et al. using an in vitro model of rat podocytes [68]; however, the precise mechanisms need to be clarified. Also, complement activation on podocytes can cause nephrin dissociation from the actin cytoskeleton with disruption of the slit diaphragm, GFB damage, and subsequent onset of proteinuria, as suggested by the Heymann nephritis (HN) model [54, 61].

Complement-mediated injury and podocyte response

Podocytes rely on several adaptive mechanisms to mitigate complement-mediated injury. Autophagy, a highly conserved mechanism of lysosome-mediated degradation of damaged organelles or nonfunctional proteins, is enhanced after sublytic complement damage in mouse podocytes, whereas its inhibition amplifies complement-mediated cell injury [69]. Liu et al. investigated the role of autophagy in PMN, comparing podocytes from PMN patients to cultured mouse podocytes exposed to sublytic complement activity, and they found impaired autophagy in podocytes from PMN patients, characterized by intracellular accumulation of p62 (marker of impaired autophagy) and increase in autophagic vacuoles [70].

Podocyte-derived VEGF has also a bivalent function, as it is described that its overexpression can cause a collapsing glomerulopathy, while its inhibition is associated with GFB disruption, proteinuria, and possible development of thrombotic microangiopathy as well [71]. The putative mechanism is that, in normal conditions, VEGF signalling can regulate complement activity on podocytes and protect them from complement-mediated injury by increasing local CFH production, while its inhibition would provoke reduced levels of CFH, and podocytes would become more vulnerable to the injury.

More recently, new interesting mechanisms have been described to protect podocytes from injury, as reported by Medica et al. using a co-culture model of glomerular endothelial cells and podocytes. In particular, they demonstrated that extracellular vesicles derived from endothelial progenitor cells and involved in intercellular crosstalk (by transferring of proteins, lipids, and genetic material) are able to protect both glomerular endothelial cells and podocytes from complement (C5a)- and cytokine-mediated injury [72]. In particular, they showed that pre-stimulation of endothelial cells with extracellular vesicles prevented podocyte apoptosis and GFB disruption, and this protective effect could be mainly secondary to RNA transfer from the extracellular vesicles to damaged endothelial cells and podocytes.

Despite a tight surveillance of the complement system, including the activity of soluble and membrane-bound regulators, together with the protective mechanisms previously described to escape the injury, unrestricted complement activation can exceed those regulatory mechanisms, causing host tissue injury, as reported in various diseases including glomerulonephritis [73], hemolytic uremic syndrome (HUS) [74], sepsis [75], systemic lupus erythematosus [76], rheumatoid arthritis [77], organ transplant rejection [78], and age-related macular degeneration [79].

Summary and conclusions

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, 80,81,82].

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, 83,84,85,86,87,88,89,90]. 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.