The current physiological concept of the slit diaphragm did emerge more than fourty years ago. Since then the SD has been regarded as the highly structured, “zipper-like” outermost part of the glomerular filtration barrier. NEPHRIN had been identified as a potential core protein, organizing the structure, function and signaling of the slit diaphragm. Our reevaluation of both murine and human null mutants for Nphs1 as well as a murine null mutant of Neph1 showed that rudimentary slit diaphragms can form in the absence of either protein. In addition our phylogenetic evaluation indicated that avian slit diaphragms are formed in the absence of NEPHRIN. Ultrastructural evaluation using cryo-electrontomography and 3D reconstruction techniques we found a multilayered protein scaffold consisting of two compartments. The lower compartment facing the glomerular basement membrane was build up by NEPH1 molecules while the upper compartment next to Bowman’s space contained NEPHRIN molecules. Our future work is directed to further elucidate the biophysical properties of this unique junctional scaffold and to precisely determine its molecular composition.