Increased glomerular capillary pressure (glomerular hypertension) and increased glomerular filtration rate (glomerular hyperfiltration) have been proven to cause glomerulosclerosis in animal models and are likely to be operative in patients. Since podocytes cover the glomerular basement membrane, they are exposed to tensile stress due to circumferential wall tension and to fluid shear stress arising from filtrate flow through the narrow filtration slits and through Bowman's space. In vitro evidence documents that podocytes respond to tensile stress as well as to fluid shear stress. Several proteins are discussed in this review that are expressed in podocytes and could act as mechanosensors converting mechanical force via a conformational change into a biochemical signal. The cation channels P2X4 and TRPC6 were shown to be involved in mechanosignaling in podocytes. P2X4 is activated by stretch-induced ATP release, while TRPC6 might be inherently mechanosensitive. Membrane, slit diaphragm and cell-matrix contact proteins are connected to the sublemmal actin network in podocytes via various linker proteins. Therefore, actin-associated proteins, like the proven mechanosensor filamin, are ideal candidates to sense forces in the podocyte cytoskeleton. Furthermore, podocytes express talin, p130Cas, and fibronectin that are known to undergo a conformational change in response to mechanical force exposing cryptic binding sites. Downstream of mechanosensors, experimental evidence suggests the involvement of MAP kinases, Ca and COX2 in mechanosignaling and an emerging role of YAP/TAZ. In summary, our understanding of mechanotransduction in podocytes is still sketchy, but future progress holds promise to identify targets to alleviate conditions of increased mechanical load.

Dedifferentiation and loss of podocytes are the major cause of chronic kidney disease. Dach1, a transcription factor that is essential for cell fate, was found in genome‐wide association studies to be associated with the glomerular filtration rate. We found that podocytes express high levels of Dach1 in vivo and to a much lower extent in vitro. Parietal epithelial cells (PECs) that are still under debate to be a type of progenitor cell for podocytes expressed Dach1 only at low levels. The transfection of PECs with a plasmid encoding for Dach1 induced the expression of synaptopodin, a podocyte‐specific protein, demonstrated by immunocytochemistry and Western blot. Furthermore, synaptopodin was located along actin fibres in a punctate pattern in Dach1‐expressing PECs comparable with differentiated podocytes. Moreover, dedifferentiating podocytes of isolated glomeruli showed a significant reduction in the expression of Dach1 together with synaptopodin after 9 days in cell culture. To study the role of Dach1 in vivo, we used the zebrafish larva as an animal model. Knockdown of the zebrafish ortholog Dachd by morpholino injection into fertilized eggs resulted in a severe renal phenotype. The glomeruli of the zebrafish larvae showed morphological changes of the glomerulus accompanied by down‐regulation of nephrin and leakage of the filtration barrier. Interestingly, glomeruli of biopsies from patients suffering from diabetic nephropathy showed also a significant reduction of Dach1 and synaptopodin in contrast to control biopsies. Taken together, Dach1 is a transcription factor that is important for podocyte differentiation and proper kidney function.

It is generally assumed that pathway-specific transcriptional activators recruit pleiotropic coactivators (such as chromatin-modifying complexes or general transcription factors), while specific repressors contact pleiotropic corepressors creating an inaccessible chromatin by the action of histone deacetylases. We have previously shown that the negative regulator Opi1 of yeast phospholipid biosynthesis inhibits transcription by recruiting corepressors Sin3 and Cyc8 in the presence of precursor molecules inositol and choline. To get access to its target genes, Opi1 physically contacts and counteracts DNA-bound activator Ino2. By using chromatin immunoprecipitation, we show that Sin3 and Cyc8 can be detected at Opi1 target promoters INO1 and CHO2 under repressing and derepressing conditions and that corepressor binding is effective even in the absence of Opi1, while Ino2 is absolutely required. Thus, corepressors may be recruited not only by repressors but also by activators such as Ino2. Indeed, we could demonstrate direct interaction of Ino2 with Sin3 and Cyc8. The Opi1 repressor interaction domain within Ino2 is also able to contact Sin3 and Cyc8. Recruitment of corepressors by an activator is not a regulatory exception as we could show that activators Pho4 and Hac1 also contain domains being able to interact with Sin3 and Cyc8.

Hypertension is one of the central causes of kidney damage. In the past it was shown that glomerular hypertension leads to morphologic changes of podocytes and effacement and is responsible for detachment of these postmitotic cells. Because we have shown that podocytes are mechanosensitive and respond to mechanical stress by reorganization of the actin cytoskeleton in vitro, we look for mechanotransducers in podocytes. In this study, we demonstrate that the extracellular matrix protein fibronectin (Fn1) might be a potential candidate. The present study shows that Fn1 is essential for the attachment of podocytes during mechanical stress. By real-time quantitative PCR as well as by liquid chromatography-mass spectrometry, we found a significant up-regulation of Fn1 caused by mechanical stretch (3 d, 0.5 Hz, and 5% extension). To study the role of Fn1 in cultured podocytes under mechanical stress, Fn1 was knocked down (Fn1 KD) by a specific small interfering RNA. Additionally, we established a Fn1 knockout (KO) podocyte cell line (Fn1 KO) by clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9). During mechanical stress, a significant loss of podocytes (>80%) was observed in Fn1 KD as well as Fn1 KO podocytes compared with control cells. Furthermore, Fn1 KO podocytes showed a significant down-regulation of the focal adhesion proteins talin, vinculin, and paxillin and a reduced cell spreading, indicating an important role of Fn1 in adhesion. Analyses of kidney sections from patients with diabetic nephropathy have shown a significant up-regulation of FN1 in contrast to control biopsies. In summary, we show that Fn1 plays an important role in the adaptation of podocytes to mechanical stress.

Glomerular hypertension causes glomerulosclerosis via the loss of podocytes, which are challenged by increased mechanical load. We have demonstrated that podocytes are mechanosensitive. However, the response of podocytes to mechanical stretching remains incompletely understood. Here we demonstrate that the actin-bundling protein fascin-1 plays an important role in podocytes that are exposed to mechanical stress. Immunofluorescence staining revealed colocalization of fascin-1 and nephrin in mouse kidney sections. In cultured mouse podocytes fascin-1 was localized along actin fibers and filopodia in stretched and unstretched podocytes. The mRNA and protein levels of fascin-1 were not affected by mechanical stress. By Western blot and 2D-gelelectrophoresis we observed that phospho-fascin-1 was significantly downregulated after mechanical stretching. It is known that phosphorylation at serine 39 (S39) regulates the bundling activity of fascin-1, e.g. required for filopodia formation. Podocytes expressing wild type GFP-fascin-1 and non-phosphorylatable GFP-fascin-1-S39A showed marked filopodia formation, being absent in podocytes expressing phosphomimetic GFP-fascin-1-S39D. Finally, the immunofluorescence signal of phosphorylated fascin-1 was strongly reduced in glomeruli of patients with diabetic nephropathy compared to healthy controls. In summary, mechanical stress dephosphorylates fascin-1 in podocytes in vitro and in vivo thereby fascin-1 may play an important role in the adaptation of podocytes to mechanical forces.