Sybille Koehler

Podocytes, epithelial cells of the glomerular filtration barrier, are constantly exposed to biomechanical forces. These include hydrostatic pressure and shear stress, which increase during diseases such as hypertension or diabetes. To sense and respond to such changes in their physical environment, podocytes express mechanosensors and mechanotransducers. To deepen our knowledge about renal mechanotransduction mechanisms, we used Drosophila nephrocytes. Nephrocytes and mammalian podocytes are highly similar in morphology and molecular make-up of the filtration barrier; thus, nephrocytes are considered the homologue cells to podocytes. In addition, nephrocytes also experience biomechanical forces because of haemolymph movement. Here, we investigated the role of the mechanotransducer Piezo in larval garland nephrocytes. Depletion of Piezo produces only a mild functional phenotype, whereas elevated Piezo levels result in a severe phenotype with functional and morphological disturbances. Increased Piezo levels also cause the accumulation of actin stress fibres, increased Cubilin expression, more acidic vesicles, increased mitochondrial mass and/or activity, and elevated superoxide levels.

Due to their position on glomerular capillaries, podocytes are continuously counteracting biomechanical filtration forces. Most therapeutic interventions known to generally slow or prevent the progression of chronic kidney disease appear to lower these biomechanical forces on podocytes, highlighting the critical need to better understand podocyte mechano-signalling pathways. Here we investigated whether the mechanotransducer Piezo is involved in a mechanosensation pathway in Drosophila nephrocytes, the podocyte homologue in the fly. Loss of function analysis in Piezo depleted nephrocytes reveal a severe morphological and functional phenotype. Further, pharmacological activation of endogenous Piezo with Yoda1 causes a significant increase of intracellular Ca⁺⁺ upon exposure to a mechanical stimulus in nephrocytes, as well as filtration disturbances. Elevated Piezo expression levels also result in a severe nephrocyte phenotype. Interestingly, expression of Piezo which lacks mechanosensitive channel activity, does not result in a severe nephrocyte phenotype, suggesting the observed changes in Piezo wildtype overexpressing cells are caused by the mechanosensitive channel activity. Moreover, blocking Piezo activity using the tarantula toxin GsMTx4 reverses the phenotypes observed in nephrocytes overexpressing Piezo. Taken together, here we provide evidence that Piezo activity levels need to be tightly regulated to maintain normal pericardial nephrocyte morphology and function.

Kidney filtration is ensured by the interaction of podocytes, endothelial and mesangial cells. Immunoglobulin accumulation at the filtration barrier is pathognomonic for glomerular injury. The mechanisms that regulate filter permeability are unknown. Here, we identify a pivotal role for the proteasome in a specific cell type. Combining genetic and inhibitor-based human, pig, mouse, and Drosophila models we demonstrate that the proteasome maintains filtration barrier integrity, with podocytes requiring the constitutive and glomerular endothelial cells the immunoproteasomal activity. Endothelial immunoproteasome deficiency as well as proteasome inhibition disrupt the filtration barrier in mice, resulting in pathologic immunoglobulin deposition. Mechanistically, we observe reduced endocytic activity, which leads to altered membrane recycling and endocytic receptor turnover. This work expands the concept of the (immuno)proteasome as a control protease orchestrating protein degradation and antigen presentation and endocytosis, providing new therapeutic targets to treat disease-associated glomerular protein accumulations.

Biological and biomedical research using Drosophila melanogaster as a model organism has gained recognition through several Nobel prizes within the last 100 years. Drosophila exhibits several advantages when compared to other in vivo models such as mice and rats, as its life cycle is very short, animal maintenance is easy and inexpensive and a huge variety of transgenic strains and tools are publicly available. Moreover, more than 70% of human disease-causing genes are highly conserved in the fruit fly. Here, we explain the use of Drosophila in nephrology research and describe two kidney tissues, Malpighian tubules and the nephrocytes. The latter are the homologous cells to mammalian glomerular podocytes and helped to provide insights into a variety of signaling pathways due to the high morphological similarities and the conserved molecular make-up between nephrocytes and podocytes. In recent years, nephrocytes have also been used to study inter-organ communication as links between nephrocytes and the heart, the immune system and the muscles have been described. In addition, other tissues such as the eye and the reproductive system can be used to study the functional role of proteins being part of the kidney filtration barrier.

Podocytes are specialized epithelial cells of the kidney glomerulus and are an essential part of the filtration barrier. Because of their position, they are exposed to constant biomechanical forces such as shear stress and hydrostatic pressure. These forces increase during disease, resulting in podocyte injury. It is likely podocytes have adaptative responses to help buffer against deleterious mechanical force and thus reduce injury. However, these responses remain largely unknown. Here, using theDrosophilamodel, we show the mechanosensor Cheerio (dFilamin) provides a key protective role in nephrocytes. We found expression of an activated mechanosensitive variant of Cheerio rescued filtration function and induced compensatory and hypertrophic growth in nephrocytes depleted of the nephrocyte diaphragm proteins Sns or Duf. Delineating the protective pathway downstream of Cheerio we found repression of the Hippo pathway induces nephrocyte hypertrophy, whereas Hippo activation reversed the Cheerio-mediated hypertrophy. Furthermore, we find Yorkie was activated upon expression of active Cheerio. Taken together, our data suggest that Cheerio acts via the Hippo pathway to induce hypertrophic growth, as a protective response in abnormal nephrocytes.

Podocyte injury is a major cause of chronic kidney disease. However, understanding podocyte‐specific responses at a systems level is difficult when glomerular cellular composition is altered by loss of podocytes or increase of extracellular matrix. We developed and applied proteome analysis to very small sample amounts to podocyte fractions isolated from single mice after induction of glomerular disease and to single glomeruli from patients with Focal segmental glomerulosclerosis (FSGS). Transcriptome and proteome analysis of glomeruli from patients with FSGS revealed an underrepresentation of podocyte‐specific genes and proteins in late‐stage disease. To circumvent this problem, we utilized two commonly used mouse models of chemically‐induced FSGS and performed proteomic analysis of pure podocyte fractions in early disease states. We observed perturbations of propagating pathways of glomerular disease such as Jak‐Stat signaling, central carbon metabolism such as TCA cycle enzymes, and mechanical stress response. Multi‐layered priorization approaches identified a co‐regulation of proteinuria with the mechanosensor protein Filamin‐B. Podocytes in human biopsies, single glomeruli from proteinuric rats and podocytes undergoing mechanical stress, showed increased expression of Filamin‐B, suggesting a role in detrimental stress responses. A nephrocyte‐specific depletion of Cher, the Filamin homolog in Drosophila, showed an altered filtration capacity while nephrocyte morphology was unaffected. This study identified conserved mechanisms of the podocyte stress response by analyzing ultrasensitive proteomes from human glomerular FSGS tissue and purified native mouse podocytes during early disease stages. The pipeline put forward here enables systematic comparisons of large‐scale proteomics data and phenotype to protein correlation. Support or Funding Information: MMR was supported by the DFG (Ri2811/1 and Ri2811/2).

Polarity signaling through the atypical PKC (aPKC)-Par polarity complex is essential for the development and maintenance of the podocyte architecture and the function of the glomerular filtration barrier of the kidney. To study the contribution of Par3A in this complex, we generated a novel Pard3 podocyte-specific knockout mouse model by targeting exon 6 of the Pard3 gene. Genetic deletion of Pard3a did not impair renal function, neither at birth nor later in life. Even challenging the animals did not result in glomerular disease. Despite its well-established role in aPKC-mediated signaling, Par3A appears to be dispensable for the function of the glomerular filtration barrier. Moreover, its homolog Pard3b, and not Pard3a, is the dominant Par3 gene expressed in podocytes and found at the basis of the slit diaphragm, where it partially colocalizes with podocin. In conclusion, Par3A function is either dispensable for slit diaphragm integrity, or compensatory mechanisms and a high redundancy of the different polarity proteins, including Par3B, Lgl, or PALS1, maintain the function of the glomerular filtration barrier, even in the absence of Par3A.