Podocytes and parietal glomerular cells: interplay through tunneling nanotubes

Background: Diabetic nephropathy (DN) causes podocyte injury leading to proteinuria. Dysfunction of subcellular organelles plays a key role in the pathogenesis of DN. Tunneling nanotubes (TNTs) are membranous channels that connect cells and allow the transfer of subcellular components, including organelles, from donor cells to damaged recipient cells. The cytosolic protein M-Sec plays a key role in the formation of TNTs between podocytes, and the M-Sec-TNT system plays a protective role in experimental DN. However, it is not known whether podocytes can form TNTs with other glomerular cell types. Aim: To study whether functionally active TNTs form between podocytes and glomerular parietal epithelial cells (PECs), which are considered podocyte precursors. Methods. Ex vivo study: M-Sec expression was studied on kidney sections obtained from patients with type 2 diabetes (DM2) and overt nephropathy and non-diabetic (ND) control subjects by immunohistochemistry (IHC). The glomerular localization of M-Sec was assessed using double immunofluorescence. In vitro study: Podocytes were exposed either to serum deprivation or diabetes-related insults (high glucose, AGEs, MCP-1) or vehicle, then co-cultured with PECs for 24h. TNT formation was assessed in cells stained with the fluorescent plasma membrane dye, WGA. Mitochondrial transfer was evaluated by monitoring transfer from PECs carrying mitochondria-GFP to recipient podocytes pre-exposed to high glucose and stained with the Cell Tracker Blue. Analysis were performed using both fluorescent and DIC microscopy. Experiments were performed using PECs; transfected with either M-Sec shRNA or a mock plasmid. Results: Ex vivo study: In patients with DN, several cells, including those located at the level of Bowman’s capsule, expressed M-Sec. Double immunofluorescence confirmed the expression of M-Sec in PECs. In vitro study: TNTs formed between PECs and podocytes pre-exposed to serum deprivation, a well-established TNT trigger. Furthermore, diabetes–related insults significantly increased the number of podocytes and PECs connected by TNTs. Finally, TNTs between podocytes and PECs were functionally active and allowed the transfer of mitochondria from PECs to podocytes pre-exposed to HG through an M-Sec-dependent mechanism. Conclusions: This study demonstrated that diabetes-related insults induce the formation of TNTs between PECs and podocytes, allowing the transfer of mitochondria towards injured podocytes.