Legend has it that Blues guitarist extraordinaire Robert Johnson sold his soul to the devil in return for being taught how to play the guitar. The deal was consummated at the crossroads of Highway 49 (known as the Blues Highway) and Route 61 in Clarksdale, Mississippi in 1927. Robert Johnson went on to become the father of American blues music, The King of the Delta Blues, spawning a generation and genre of African-American Blues musicians. It remains to be determined whether the podocyte in being positioned also at the crossroads of innate and adaptive immunity, was by divine intervention, or perhaps a deal made with the devil, similar to that of Robert Johnson. This is of more than passing interest, since it may well relate to podocyte longevity and lifetime/survival. You see, Robert Johnson died at the age of 27, starting a sad legacy of rock musicians who did not make it past their 27th birthday (see Supplemental Appendix). In a similar fashion, the podocyte may live out its functional existence in a normal fashion, or if it is involved in a disease process, for example focal segmental glomerulosclerosis (FSGS), may die young, the equivalent of an unfortunate 27 year-old rock musician. Please indulge this metaphysical conceit (with appreciation to John Donne), placing glomerular podocytes involved in FSGS in a position similar to doomed rock musicians. It is particularly fitting that it was Robert Johnson who wrote the song "Crossroads" (originally "Cross Road Blues"), emphasizing for us the intersection of two key immune pathways. Our mission is to determine how best to protect, preserve, and/or restore the life of our kidney rock star, the podocyte. 
Focal segmental glomerulosclerosis (FSGS) is a common glomerular disorder that manifests clinically with nephrotic syndrome and > 80% foot process effacement on electron microscopy. FSGS accounts for about 20% of cases of nephrotic syndrome in children and 40% in adults, and is the most common glomerular disorder leading to end stage kidney disease (ESKD) in the United States with a prevalence of 4%. Progression to ESKD occurs in 40-60% of patients within 10-20 years from diagnosis, making this the most common primary glomerular disease leading to dialysis in the United States. Despite many clinical trials, no effective treatment has been identified. Following transplantation, recurrence of FSGS occurs in 20-50% of adults and up to 80% in high-risk pediatric patients. Risk factors for recurrent FSGS include younger age (children less than 6 at onset), non-black race, rapid progression to ESKD (less than 3 years from diagnosis), severe proteinuria immediately prior to transplantation, and the loss of a previous allograft(s) to recurrence. Recurrence of FSGS increases the risk of renal dysfunction and early graft loss.
Recurrence of FSGS can develop rapidly after transplantation, sometimes within minutes to hours, suggesting the presence of a circulating factor(s) that could mediate an early injury to the glomerulus. Further evidence includes the following: 1) plasmapheresis can induce remission of proteinuria, and 2) serum from patients with FSGS induces proteinuria in rats, and alters albumin permeability in isolated glomeruli. Moreover, in two clinical reports, living donor kidney transplant recipients who experienced severe recurrent FSGS, underwent transplant nephrectomy with re-transplantation of the affected kidney into a non-FSGS recipient resulting in rescue of the donor kidney function with resolution of proteinuria, providing further evidence for a circulating factor(s). Several review articles provide details supporting the existence of circulating factors that are beyond the scope of this article.
Despite the lack of evidence of immune infiltrates in human FSGS biopsy samples, an immune component has been thought to contribute to FSGS. For example, the identification of IgM and C3 in glomerular deposits which was associated with both unfavorable response to therapy and worse renal outcomes supports this concept. T-cell involvement has been described in the Buffalo/Mna rat model of FSGS recurrence after renal transplantation, and in human recurrent FSGS needle aspirate samples. However, neither B cell nor T cell infiltrates are consistently found in FSGS. Nonetheless, immunosuppression, which was associated with reduced proteinuria in the Buffalo/Mna rat model, has been the main therapy in humans after plasmapheresis or plasma absorption. The most studied immune-directed therapies have included corticosteroids, inhibitors of T lymphocyte calcineurin (cyclosporine A and tacrolimus), alkylating agents (cyclophosphamide and chlorambucil), and mycophenolate mofetil. A number of case reports and non-controlled series have reported benefit from rituximab but this has not been universally successful. Similarly, it remains unclear if the benefit of these immunosuppressive agents is linked to immunomodulatory functions or to direct podocyte effects.
The glomerular capillary wall consists of three layers: 1) the fenestrated endothelial cell, 2) the glomerular basement membrane (GBM), and 3) the podocyte, the last barrier to prevent loss of protein into Bowman's space. Thus, the podocyte is anatomically located in a position to be a sentinel for the kidney's innate immune system. The mammalian innate immune system was originally intended to protect its host from outside pathogens, for example viruses, bacteria and fungi - pathogen associated molecular patterns (PAMP's) and in the process distinguish self from non-self. The theory of immunity has since evolved to the recognition of danger signals proposed by Polly Matzinger and more recently to the discontinuity theory: that immune activation can be induced by changes (any) in the environment or the cell rather than by detection of specific, defined molecules. Ultimately, the innate immune response requires a balance between the appropriate degree of aggression in the face of a serious pathogenic threat, but also the ability to limit local tissue damage with a controlled response.
Podocytes have been shown to express Toll-like receptors (TLRs), which recognize PAMPs, and certain endogenous signals, for example cellular breakdown products (Damage-Associated Molecular Patterns- DAMPs), e.g. reactive oxygen species, mitochondrial or, endoplasmic reticulum stress, DNA, RNA, nucleic acid fragments, etc.. TLRs which bind to DAMP's/PAMP's are part of the cellular (host) innate immune defense system, which, once activated, induce secretion of specific cytokines and chemokines that prompt the recruitment of other components of the innate immune response. TLR stimulation activates nuclear factor Kappa B (NF Kappa B) in podocytes, and key components of TLR signaling including MyD88, IRAK, TRAF 6, etc. ( Figure 1 ). It has been proposed that the podocyte's role in innate immunity may predispose it to injury, depending on chronicity.
This is supported by the demonstration that TLR stimulation leads to podocyte apoptosis, and that blocking this pathway may prevent the progression of fibrosis and disease. In addition to TLRs there are a number of other important Pattern Recognition Receptors (PRRs) that are expressed by podocytes. These include the major components of the inflammasome, Nod-like receptors (NLRP3), ASC and caspase 1. The inflammasome is an intracellular protein complex involved in innate immune function including autophagy, apoptosis, fibrosis and secretion of pro-inflammatory cytokines including IL1-Beta and IL18. Podocytes also express retinoic acid-inducible gene 1 (RIG-1) that belongs to the family of RIG-I-like helicases that recognize viral RNA. Other family members include MDA5 and upon activation by recognition of viral RNA by cytoplasmic sensors, signal to the mitochondrial adaptor MAVS inducing an Interferon-regulatory factor (IRF)3, and IRF7-mediated type 1 IFN response ( Figure 1 ).
Recently, the cGAS-STING axis has been identified as playing an important role in podocyte innate.
Immunity. cGAS, a PRR, is considered to be a key sensor of cytosolic DNA leading to catalyzation of second messenger 2'3'-cGAMP, which binds to the endoplasmic reticulum membrane adaptor STING (encoded by TMEM173) leading to activation of TANK-binding kinase 1 (TBK1), which in turn activates nuclear interferon regulatory factor 3 (IRF3) and leads to the production of type I interferons ( Figure 1 ). The development of the cGAS-STING cytoplasmic defense system provides recognition of foreign nucleic acids, including pathogens/PAMPs, and DAMPs, as well as self-mitochondrial DNA leakage, cellular stress, and senescent break down products. This remarkably complex and efficient system based on the spatial coordination of DNA induced-cGAS-STING activity allows for antiviral immunity and balanced innate immune responses toward self-DNA. While the crucial roles of the cGAS-STING pathway in immune cells and the immune defense have been extensively investigated, its function in non-immune cells, such as podocytes, remains largely unknown. It is not known if STING contributes to the development and/or progression of glomerular diseases, however, our group has been investigating this, and our work is summarized in this article.
Beyond its role in innate immunity, the podocyte is capable of contributing to the adaptive immune response. The podocyte has been shown to possess properties of non-hematopoietic professional antigen-presenting cells (APCs). Expression of both class I MHC antigens and, under certain inflammatory conditions, MHC class II antigens, contributing to the activation of CD8 and CD4 positive T cells, respectively, has been demonstrated on the podocyte. Moreover, podocytes express B7-1 (also known as CD80), a co-stimulatory molecule for T cells which is inducible under conditions of stress and was first reported in the context of the nephrotic syndrome. B7-1/CD80 is part of the B cell and APC repertoire and provides the second signal to T cells that allows amplification of the response to peptide antigens presented in the context of the appropriate MHC class I/II background (signal 1). B7-1 binds to CD28 on the T cell membrane, resulting in a positive co-stimulatory signal (signal 2). Interestingly, B7-2 (CD86), a co-stimulatory molecule presents on B cells and APCs that is also capable of binding to CD28 on T cells, is not present on podocytes.
The podocyte is in a position to integrate and act upon the range of immune signals, from T/B cell secreted cytokines to DAMPs/PAMPs. When podocytes are activated, through TLR's, cGAS-STING, or B7-1, etc., the change, mediated through the actin cytoskeleton, can affect the slit diaphragm proteins, allowing the passage of protein (proteinuria) or potentially danger signals, e.g. pathogens, into Bowman's space, and hence into the urine and excreted. This potential for the excretion of danger/pathogen imbues the kidney, by virtue of the podocyte, with the capacity for immune defense mediated through a combination of innate and adaptive immune mechanisms. Importantly, clearance of danger into the urine avoids the activation of inflammatory mediators/cytokines that could result in unnecessary local tissue damage.