Advanced electron microscopic techniques provide a deeper insight into the peculiar features of podocytes.

Podocytes constitute the outer layer of the glomerular filtration barrier, where they form an intricate network of interdigitating foot processes which are connected by slit diaphragms. A hitherto unanswered puzzle concerns the question of whether slit diaphragms are established between foot processes of the same podocyte or between foot processes of different podocytes. By employing focused ion beam-scanning electron microscopy (FIB-SEM), we provide unequivocal evidence that slit diaphragms are formed between foot processes of different podocytes. We extended our investigations of the filtration slit by using dual-axis electron tomography of human and mouse podocytes as well as of Drosophila melanogaster nephrocytes. Using this technique, we not only find a single slit diaphragm which spans the filtration slit around the whole periphery of the foot processes but additional punctate filamentous contacts between adjacent foot processes. Future work will be necessary to determine the proteins constituting the two types of cell-cell contacts.

Polycystin-2 takes different routes to the somatic and ciliary plasma membrane.

Polycystin-2 (also called TRPP2), an integral membrane protein mutated in patients with cystic kidney disease, is located in the primary cilium where it is thought to transmit mechanical stimuli into the cell interior. After studying a series of polycystin-2 deletion mutants we identified two amino acids in loop 4 that were essential for the trafficking of polycystin-2 to the somatic (nonciliary) plasma membrane. However, polycystin-2 mutant proteins in which these two residues were replaced by alanine were still sorted into the cilium, thus indicating that the trafficking routes to the somatic and ciliary plasma membrane compartments are distinct. We also observed that the introduction of dominant-negative Sar1 mutant proteins and treatment of cells with brefeldin A prevented the transport into the ciliary plasma membrane compartment, whereas metabolic labeling experiments, light microscopical imaging, and high-resolution electron microscopy revealed that full-length polycystin-2 did not traverse the Golgi apparatus on its way to the cilium. These data argue that the transport of polycystin-2 to the ciliary and to the somatic plasma membrane compartments originates in a COPII-dependent fashion at the endoplasmic reticulum, that polycystin-2 reaches the cis side of the Golgi apparatus in either case, but that the trafficking to the somatic plasma membrane goes through the Golgi apparatus whereas transport vesicles to the cilium leave the Golgi apparatus at the cis compartment. Such an interpretation is supported by the finding that mycophenolic acid treatment resulted in the colocalization of polycystin-2 with GM130, a marker of the cis-Golgi apparatus. Remarkably, we also observed that wild-type Smoothened, an integral membrane protein involved in hedgehog signaling that under resting conditions resides in the somatic plasma membrane, passed through the Golgi apparatus, but the M2 mutant of Smoothened, which is constitutively located in the ciliary but not in the somatic plasma membrane, does not. Finally, a dominant-negative form of Rab8a, a BBSome-associated monomeric GTPase, prevented the delivery of polycystin-2 to the primary cilium whereas a dominant-negative form of Rab23 showed no inhibitory effect, which is consistent with the view that the ciliary trafficking of polycystin-2 is regulated by the BBSome.

Ultrastructural insights in the interface between generated renal tubules and a polyester interstitium.

In regenerative medicine, stem/progenitor cells are emerging as potential candidates for the treatment of renal failure. However, the mechanism of regeneration of renal tubules from stem/progenitor cells is not well-elucidated. In this study, a new method was developed for the generation of tubules replacing coating by extracellular matrix proteins. Renal stem/progenitor cells are mounted between layers of polyester fleece. This artificial interstitium supports spatial development of tubules within 13 days of perfusion culture in chemically defined Iscove's modified Dulbecco's medium (IMDM) containing aldosterone as the tubulogenic factor. Whole mount label by soybean agglutinin (SBA) showed that generated tubules exhibited a lumen and a continuously developed basal lamina. Immuno-labeling for cytokeratin Endo-A demonstrated the presence of isoprismatic epithelial cells, and laminin gamma1, occludin, and Na/K-ATPase alpha5 labeling revealed typical features of a polarized epithelium. To get first insight in the interface between tubules and polyester interstitium, transmission electron microscopy (TEM) was performed. The results showed that the generated tubules exhibited polar differentiation with a continuously developed basal lamina consisting of a lamina rara interna, lamina densa, and lamina rara externa. Collagen type III was found to be the linking molecule between the basal lamina and the surrounding polyester fibers by immuno labeling studies. Thus, the findings demonstrate that the spatial development involves the interface between the tubular basal lamina and the polyester interstitium of tubules and is not restricted to the epithelial portion.