This study is the first to demonstrate that B7-1 is expressed in podocytes under a variety of stress conditions. Moreover, the results of this study establish the clinical significance of podocyte B7-1 expression and elucidate its role in the pathogenesis of nephrotic syndrome. We specifically demonstrate that podocyte B7-1 reorganizes the actin cytoskeleton of podocytes and modulates SD organization. The effects are independent of T cells and B cells and represent a novel unique function of B7-1. This study also is the first to show that LPS injection in mice induces B7-1–dependent nephrotic syndrome with FP effacement and urinary protein loss.
So far, B7-1 had been assumed to be predominantly expressed on B cells and other professional APCs. The traditional role of B7-1 is T cell costimulation (13
). Along with the interaction of the T cell receptor with antigen-MHC complexes, the interaction of B7 with its receptors CD28 and CTLA-4 on T cells plays a pivotal role in T cell–mediated immunity (13
). In T cells, binding of B7-1 to its receptors on the T cell surface triggers migration of protein kinases, actin-binding proteins, and other accessory molecules to the T cell–APC interface (40
). This formation of the immunological synapse causes a rearrangement of the T cell actin cytoskeleton (40
), activates protein tyrosine kinases that couple to PI3K, and controls the activity of Ras- and Rho-family GTPases in T cells (45
The role of B7-1 in T cell costimulation is well established and defines a dialogue between a B7-1–expressing APC and a T cell to which the costimulatory signal is conveyed (13
). In contrast, B7-1–mediated outside-in signaling in B7-1–expressing APCs is still obscure. With respect to outside-in signaling through B7-1 on B7-1–expressing cells, two studies have been published. One study showed that cross-linking of B7-1 on B lymphoma Raji cells induces tyrosine phosphorylation of intracellular proteins, indicating a role of B7-1 in outside-in signaling (46
). Further support for a role of B7-1 in outside-in signaling comes from a recent report showing that CTLA-4-Ig–mediated ligation of B7-1 regulates tryptophan catabolism in dendritic cells (47
). These changes are reminiscent of the changes seen in SD protein redistribution after ligation/activation of B7-1 in podocytes (Figure ).
In human keratinocytes, B7-1 gene expression is induced by allergens and irritants (16
). In P815 mastocytoma tumor cells, B7-1 gene expression is induced by oxidative stress (17
). In APCs, MyD88-independent LPS/TLR-4 signaling is the strongest activator of B7-1 expression (20
). Similarly, B7-1 induction in podocytes appears to be downstream of various signaling pathways, including TLR signaling (Figure ). Generally, somatic cells grown in culture are, for the most part, quite indifferent to LPS (20
), and earlier studies had shown that macrophages are the primary target cells of LPS in septic shock (20
). Here we demonstrate that podocytes constitutively express the LPS receptor TLR-4 and its coreceptor CD14 and respond to LPS exposure with upregulation of B7-1. This is another unanticipated finding of the present study.
The LPS experiments (Figures and ) allowed us to explore several important questions. First, they established that the induction of podocyte B7-1 in conditions with FP effacement and proteinuria was causative and not the consequence of the nephrotic condition, since B7-1–/– mice were protected from LPS-induced proteinuria. Second, one might argue that the effects of LPS are predominantly mediated through the action of T cells. Our data refute this hypothesis, because (a) SCID mice lacking T and B cells showed the same degree of FP effacement and proteinuria as WT mice; (b) exposure of cultured podocytes in the absence of any other cells was sufficient to upregulate B7-1 and reorganize the podocyte actin cytoskeleton; and (c) the cell-free microbead-mediated cross-linking of B7-1 on α3–/– podocytes was sufficient to redistribute vital SD proteins. Collectively, these data suggest that B7-1 expression by podocytes represents a pathogenic mechanism for disruption of the glomerular filtration apparatus that is independent of T cell activation.
An intriguing question remains, why do podocytes upregulate B7-1? In the grass shrimp, Palaemonetes pugio
, podocytes are involved in the ultrafiltration of hemolymph where micro- and macromolecular substances are cleared (49
). In the pre-molt stage of this shrimp, podocyte assemblages extend into the efferent hemolymph channels and are in direct contact with immune cells, and they may serve as a component of the LPS-sensing (20
) innate immune response. The upregulation of podocyte B7-1 by LPS may be a phylogenetically preserved mechanism, which can be reactivated under pathological conditions, e.g., in patients with Gram-negative sepsis. Consistent with this idea, the development of transient proteinuria has been found during the course of Gram-negative sepsis (50
). Moreover, a prospective study showed that in postoperative septic patients microalbuminuria is an early indicator of increased glomerular permeability, but not in nonseptic patients (51
). The resulting, transient, proteinuria may be a physiological response aimed at clearing the circulation from pathogen-associated molecules. In human minimal-change disease, the onset of nephrotic syndrome is often preceded by an infection or allergic reaction (52
). This raises the intriguing possibility that nephrotic syndrome in minimal-change disease represents the persistence of a normally beneficial response caused by genetic defects in the B7-1 pathway or by other unphysiological activators. The sustained B7-1 activation in podocytes, in turn, results in continuing nephrotic syndrome.
In summary, we have demonstrated a novel function for B7-1 in the pathogenesis of proteinuria that is distinct from its role in costimulation (Figure ). Our findings suggest a novel function for B7-1 in danger signaling by podocytes. The B7-1 knockout by itself is not conclusive since it is not podocyte specific. While the SCID mice control for T or B cell–mediated effects, it is still possible that dendritic cells or other APCs might contribute to the observed effects. Future studies with a podocyte-specific B7-1 knockout should allow us to address this question.
Figure 7 Pathways leading to B7-1_mediated proteinuria. Various stimuli may lead to podocyte B7-1 induction. These include genetic stimuli (e.g., deletion of α3 integrin or nephrin), toxic stimuli (PAN-induced reactive oxygen species [ROS]), (more ...)
We propose that transient B7-1–induced nephrotic syndrome may be a physiological response. Clearly, future studies will be required to explore this hypothesis in detail, but it could potentially explain how a normally beneficial danger-sensing mechanism can have deleterious effects in certain genetic backgrounds. Understanding the role of B7-1 in podocytes should allow new avenues toward the development of novel, selective podocyte-protective therapies that modulate danger signaling in podocytes and tackle proteinuria. It will be interesting to see whether B7-1–dependent pathways are also operative in chronic kidney disease.