In this study, we investigated the role of the protease CatL and the large GTPase dynamin in healthy and diseased kidney podocytes. We show that dynamin is normally required to maintain the ultrafiltration barrier in kidneys, possibly via regulation of the actin cytoskeleton in podocytes. Using a mouse model for inducible kidney failure, we found that cleavage of dynamin by a cytoplasmic form of CatL leads to podocyte failure and proteinuria. Gene delivery of CatL-resistant dynamin mutants can prevent and even reverse proteinuria. Our data define a new role for cytoplasmic CatL (trigger) and dynamin (target) in the pathogenesis of glomerular proteinuria and open new potential avenues for pharmacological intervention of kidney disease.
Previous inhibitor studies suggested that CatL is involved in proteinuric kidney disease (4
), and our current (Figure D) and previous work (3
) has shown that CatL is upregulated in rodent models for nephropathy. Consistent with these findings, we observed an increase in levels of glomerular CatL
mRNA in a number of human proteinuric diseases (Figure A) that correlated with a dramatic increase in the protein level in glomeruli (Figure C). Using knockout mice, we provide definitive evidence that CatL is essential to cause nephropathy in response to LPS in a mouse model (Figure , D–F). Furthermore, we have shown that after LPS treatment, CatL appears in the cytoplasm, due to expression of the short form of the protein. Together, the data indicate that localization of CatL to the cytoplasm represents a key event in the induction of glomerular kidney disease. Consistent with this model, we show that dynamin, a cytoplasmic protein, is essential for kidney function and that it is cleaved by CatL after LPS treatment. Importantly, the dynamin p40 fragment generated by CatL cleavage functions as a dominant-negative inducer of proteinuria. Thus, the activity of the cytoplasmic CatL, in contrast to its lysosomal counterpart, seems to be specific and to yield a functional product (p40). These data argue that complete destruction of dynamin is not necessarily required to cause kidney failure.
Intriguingly, the CatL cleavage site is highly conserved among dynamin family members (Figure A), and its accessibility is suppressed by dynamin oligomerization. Moreover, cytoplasmic CatL targets only the GTP-bound form of dynamin, implying that only a portion of dynamin is cleaved in vivo. Our previous experiments indicate that dynamin:GTP is the active form of the enzyme (19
), so it is tempting to speculate that CatL functions to switch off dynamin much like the result of dynamin self-assembly. It remains to be seen whether this novel switch-off mechanism is ever used during normal cell physiology.
EM revealed 2 populations of actin in FPs of podocytes; one is the actin bundle running above the level of slit diaphragms, and the other is the cortical actin network located beneath the plasmalemma (27
). Our data show that dynamin colocalizes with both of these populations (Figure B). It appears that FPs have the molecular makeup for constant morphological rearrangement in order to accommodate glomerular filtration. This membrane reshaping is most likely driven by rapid changes in the actin network (28
), and our study suggests that it requires functional dynamin. On its C terminus, dynamin contains a proline-arginine rich domain (PRD) that binds directly to the Src homology 3 (SH3) domains of multiple proteins, some of which are actin-regulating or -binding proteins including profilin, Nck, Grb2, syndapin, intersectin, cortactin, mABP1, and tuba (reviewed in ref. 13
). It is through these interactions that dynamin is thought to coordinate membrane remodeling and actin filament dynamics during endocytosis, cell morphogenesis, and cell migration. Despite these links between dynamin and actin, it is still unclear how exactly dynamin regulates actin dynamics. Our results identify podocytes, with their complex actin dynamics, as an appropriate cell type for investigating the interplay between dynamin’s role in endocytosis and its ability to regulate the actin cytoskeleton. Indeed, formation of a cortical actin cytoskeleton in cells expressing dynK44A
or p40 or that have been treated with LPS (Figure B) could result from the inhibition of endocytosis, misregulation of actin dynamics, or both. While at present we have no direct evidence for a direct connection between actin and dynamin, addition of LPS does not inhibit endocytosis in cultured podocytes (Supplemental Figure 6), suggesting that loss of endocytosis per se is not sufficient to generate a cortical actin network. In agreement with this conclusion are experiments showing that expression of a known endocytosis inhibitor, dominant-negative auxilinH875Q
), impaired endocytosis to the same extend as dynK44A
(Supplemental Figure 6A), yet it did not alter actin morphology (Supplemental Figure 6B). In addition, nondifferentiated podocytes contained predominantly cortical actin (Supplemental Figure 6B), yet they exhibited WT levels of endocytosis (Supplemental Figure 6A). In sum, there seems to be no correlation between levels of endocytosis and actin dynamics in podocytes, raising the possibility that dynamin mutants might primarily act on podocyte actin dynamics. This conclusion is also in line with the observation that CatL reduced endogenous dynamin by only 30% (Figure D), possibly maintaining dynamin’s role in endocytosis. Regardless of these observations, dynamin is clearly present on clathrin-coated pits in podocytes (Figure A), and changes in endocytosis have been implicated in some forms of podocyte failure (30
). Clearly, more work is needed to define the exact role(s) of dynamin in podocytes.
Proteinuria was resolved by expression of dynR725A
, and to some degree dynWT
. While our study shows that dynamin is efficiently delivered and expressed in podocytes, it is unlikely that every podocyte received dynamin. We propose that damaged podocytes may be particularly amenable to uptake of DNA presented in polymers (8
). Alternatively, it is conceivable that the effects of gene-delivered dynamin has multiple effects in podocytes. For example, it seems possible that restoration of podocyte actin has a salutary effect on the slit diaphragm function. Finally, gene-delivered dynamin in podocytes may have a non–cell-autonomous effect on other resident glomerular cells such as glomerular endothelial cells. Whatever the precise mechanism, the use of dynamin mutants as a treatment for human kidney disease is an attractive subject for future experiments.