Over two decades of research has provided interesting insights into the structure and function of the glomerular capillary loop (29
). More recent studies have addressed the relative role of podocyte versus basement membrane proteins in maintaining the permeability barrier (24
). The paper on Neph1 by Donoviel et al. (32
) describing nephrotic syndrome at birth in Neph1 mutant mice provides conclusive evidence that Neph1 plays a significant role in the normal development and function of the glomerular capillary loop. The paper by Sellin et al. (15
) describes the interaction of Neph1 with podocin in vitro, the distribution of Neph1 in a glomerular capillary loop pattern, and the transactivation of AP-1 in the presence of Tec kinases. Our current study provides conclusive evidence that Neph1 is a slit diaphragm protein, as shown by immunogold localization in the slit diaphragm.
The coimmunoprecipitation studies using anti-Neph1, anti-nephrin, and anti-FLAG Ab’s with glomerular extracts and recombinant proteins were highly suggestive of an interaction between Neph1 and nephrin at the level of their slit diaphragm spanning extracellular segments. We therefore decided to disrupt this interaction using an in vivo approach. The occurrence of complement- and leukocyte-independent proteinuria by a combination of anti-Neph1 and anti-nephrin Ab’s in individually subnephritogenic doses is highly suggestive that this interaction exists in vivo, and is at least partially altered by this Ab combination approach. Moreover, the induction of heterologous-phase proteinuria by the combination of anti-Neph1 and anti-nephrin was not associated with foot process effacement or difference in the mRNA expression of ZO-1, nephrin, and Neph1 between proteinuric and nonproteinuric subgroups, indicating that it largely affected slit diaphragm permeability due to altered protein-protein interaction.
It is important to note that the dose-response curves performed as a prelude to these studies were intended to identify an optimum subnephritogenic dose of individual Ab’s, and not to study the full extent of the nephritogenic potential of the individual Ab’s. Indeed, both anti-Neph1 and anti-nephrin start increasing slit diaphragm permeability individually (compared with preimmune serum controls) at a dose of 500 μl in rats. It is possible that future experiments directed toward investigating the nephritogenic potential of these individual Ab’s may show more proteinuria at higher doses.
The choice of an in vivo approach to test the Neph1-nephrin association over an in vitro approach using monolayers of cultured glomerular epithelial cells was made for several reasons. The slit diaphragm is the final barrier that restricts passage of plasma proteins into the urine (29
). We have now known for several years that certain Ab’s directed against podocyte proteins injected intravenously induce complement- and leukocyte-independent proteinuria (5
). We therefore rationalized that Ab’s directed against the extracellular segments of slit diaphragm proteins would bind their targets and induce slit diaphragm dysfunction that would be detectable as proteinuria. The classic example of this is mAb 5-1-6, which induces proteinuria in rats in certain doses after binding to an epitope on the extracellular segment of nephrin. An in vitro approach to this issue would be less than ideal, since there are no cultured glomerular epithelial cells that form foot processes and slit diaphragms with morphological similarity to their in vivo counterparts. In addition, cultured cells tend to form tight junctions, which are not seen in differentiated podocytes and would make the interpretation of monolayer permeability data more difficult.
The association of ZO-1 with the cytoplasmic tail of Neph1, and the interaction of nephrin with Neph1 in the slit diaphragm, also offer valuable insights into changes in podocyte proteins during proteinuria. The study complements the astute observation of Kawachi et al. (24
) that the induction of proteinuria with mAb 5-1-6, an anti-nephrin Ab, results in reduced amounts of glomerular ZO-1 by immunostaining and Western blot. In the anti-Neph1/anti-nephrin model discussed above, glomerular ZO-1 was unchanged compared with controls when rats were injected with anti-nephrin at a dose that did not cause proteinuria. Some reduction of glomerular ZO-1 content was noted with the injection of anti-Neph1 alone, testifying further to the direct association of ZO-1 with Neph1. The most dramatic reduction, however, was observed when the Ab combination induced heterologous-phase proteinuria. This indicates that the interaction between nephrin and Neph1 helps to partially stabilize the association between Neph1 and ZO-1, and that disruption of this association results in a rapid reduction of glomerular ZO-1. This allows us to postulate that a nephrin/Neph1/ZO-1 macromolecular complex exists in the slit diaphragm and may be altered in some forms of proteinuria. Whether our anti-nephrin Ab given alone in higher doses reduces glomerular ZO-1 will be a subject of future investigations designed to examine the nephritogenic phase of this Ab. It would also be interesting to conduct similar studies with mAb 5-1-6.
A substantial amount of novel data presented in the Results section allows us to propose further refinements in the organization of Neph1 molecules in the slit diaphragm. The existence of Neph1 homodimers and multimers, the coimmunoprecipitation of nephrin with native Neph1, the selective coimmunoprecipitation of nephrin with the recombinant extracellular Neph1, and the induction of complement- and leukocyte-independent heterologous-phase proteinuria by the combination of anti-nephrin and anti-Neph1 Ab’s in subnephritogenic doses all help in generating possible configurations. Neph1 homodimers/multimers may be arranged side by side in the slit diaphragm or adhere to each other from adjacent foot processes. It is also likely that Neph1 forms additional heterodimers with nephrin molecules either side by side or approaching each other from adjacent foot processes. Even though both the homodimeric association of Neph1 and a heterodimeric association with nephrin are both likely to be important in maintaining the permeability characteristics of the slit diaphragm, the occurrence of proteinuria after treatment with subnephritogenic combinations of Ab’s rather than with individual Ab’s would suggest that the latter may be more significant. In addition to the data presented above, the likelihood of a Neph1-nephrin interaction is also supported by the interaction in Drosophila
between Hibris and Dumbfounded, two proteins with structural similarity to nephrin and Neph1, respectively, during myoblast fusion (35
). Also, an association between nephrin and Neph1 has very recently also been shown by two other studies (27
We have not as yet investigated the potential interactions of Neph1 or nephrin with FAT, a member of the cadherin superfamily that also spans the slit diaphragm (11
). Interestingly, FAT also has a PDZ-binding motif that was not shown to bind ZO-1 in a recent paper also addressing the association between Neph1 and ZO-1 (28
). Moreover, the data appears to suggest that very large cadherins, such as the FAT family members, may not be involved in adhesion at all, but may have a sensing role (37
). Also, two genes related to Neph1, named Neph2
, have now been shown to be expressed in human kidney cortex by RT PCR (15
). Precise cellular and subcellular localization is still pending. Further studies are required to investigate other protein-protein associations in the slit diaphragm that may also be important in maintaining the permeability status of this important structure.