In addition to our appreciation of podocytes’ role in CKD, our view of their role in maintaining the kidney filtration barrier is dramatically changing. In vitro studies using cultured podocytes as well as the most recent two-photon microscopy of podocytes in live mice and rats (13
) have led us to regard the kidney filtration barrier as a highly dynamic structure. There is a consensus in the field that regardless of the original cause of podocyte injury (genetic, environmental, or both), podocyte response to injury follows a similar path. Specifically, injury results in FP effacement (loss of membrane extensions) driven by the global reorganization of the actin cytoskeleton, which is accompanied by proteinuria. If these early structural changes within podocytes are not reversed, progressive and severe damage occurs, which can include podocyte detachment, obliteration of the urinary space, development of segmental glomerulosclerosis, and end-stage renal failure. Thus, the current working hypothesis suggests not only that the early stages of FP effacement are reversible, but also that the reversal of effacement is critical for maintenance of healthy podocytes within the glomerulus. This dynamic model is supported by classical experiments using perfusion of rat kidneys with the polycation protamine sulfate, where FP effacement and accompanying proteinuria develop within a few minutes, yet can be fully reversed within 15 min by the administration of the polyanion heparin (14
). These rapid changes of podocyte FP architecture require FPs to retract or elongate, hence to “move” quickly along the glomerular basement membrane.
Building on this observation, we and others are using cultured podocytes to study their motility patterns in vitro (15
). Stimuli that lead to the development of FP effacement in vivo (e.g., lipopolysaccharide or puromycin aminonucleoside) have been found to cause hypermotility of cultured podocytes in cell migration assays (15
). Based on these observations, the term podocyte motility has been coined, referring to the dynamic reorganization of the interdigitating FP structure in vivo and to the cell migration of cultured podocytes in vitro, which is seen as a surrogate for the former.
Initially, it was believed that a stationary podocyte phenotype reflects a stable FP structure with intact slit diaphragms, whereas hypermotility would manifest as FP effacement in vivo (8
). This hypothesis was recently directly tested in mouse strains overexpressing either dominant active (Da) or dominant negative (Dn) mutants of RhoA (18
). In cells, expression of the Da mutant, which lasts longer in the GTP-bound state, resulted in an increase in the number of focal adhesions (FAs) and stress fibers, which in podocytes in vitro is associated with diminished motility. In contrast, expression of the Dn mutant, which cannot bind GTP, results in dramatic loss of stress fibers and FAs, and these losses lead to increased motility in podocytes in vitro. Based on the podocyte motility hypothesis, expression of Dn-RhoA specifically in podocytes was expected to induce FP effacement and proteinuria (due to increased motility), whereas expression of Da-RhoA was not predicted to have any profound phenotype based on the assumption that decreased motility in vitro equals stable FP in the glomerulus. Contrary to the podocyte motility hypothesis, expression of both these RhoA mutants resulted in FP effacement and proteinuria, demonstrating that hypermotility in vitro can lead to FP effacement and proteinuria as quickly and directly as hypomotility. Thus, the model, in which prominent stress fibers in cultured podocytes reflect intact FPs in vivo, whereas the loss of actin stress fibers and reorganization into cortical actin in vitro equals FP effacement in vivo, is somewhat oversimplified. This RhoA study demonstrated that the actin cytoskeleton in podocytes is tightly regulated and that dysregulation (either through gain- or loss-of-function mutations) has a direct consequence on the structure and thus the function of FPs. Researchers often use simplified concepts to model complex biological processes, but in the case of cultured podocytes, simplification might be misleading: e.g., an increase in actin stress fibers in vitro can be interpreted as a sign of podocyte damage; however, a decrease in stress fibers can be interpreted similarly.
The above findings also suggest a need to redefine the roles of signaling GTPases in podocytes. For example, currently, some researchers believe that the RhoA signaling pathway results in a stationary (healthy) podocyte phenotype, whereas activity of Rac1/Cdc42 underlies the motile (effaced) phenotype (8
). It seems more probable that signaling by all three members of the small Rho-type family of GTPases is highly coordinated within podocyte FPs in a manner similar to that in cultured mouse embryonic fibroblasts (19
). In addition, signaling by RhoA, Rac1, and Cdc42 has been implicated in a broad variety of other cellular processes including adhesion, proliferation, and apoptosis (20
). Thus, the effects of overexpression of mutants of these GTPases in podocytes may be mediated by processes other than the reorganization of the actin cytoskeleton in FPs.
Because interpretation of the actin dynamics in cultured podocytes does not seem to be as straightforward as originally assumed, we suggest a concept of “podocyte plasticity.” Podocyte plasticity implies that podocytes have an inherent ability to efface as well as to reform their FPs, which is driven by the global reorganization of the actin cytoskeleton and the coordinated interplay between signaling GTPases. One of the major unanswered questions with respect to the development of novel therapeutics is the critical period of plasticity for effaced (injured) podocytes. The concept of podocyte plasticity also suggests two novel druggable pathways for proteinuric kidney diseases: outside-in signaling to the actin cytoskeleton and direct regulation of the actin cytoskeleton.