Marco conceptual

To investigate the role of miRNAs in glomeruli and podocytes, three main model systems can be used: immortalized cell lines in cell culture, podocytes isolated freshly from mice and manipulations inside the murine organism. In mammalian organisms, podocytes are placed in a very specific environment. In vivo, they cover the capillary of the glomerulus. The renal filtration barrier is composed of three layers: the fenestrated endothelium including the glycocalyx, the glomerular basement membrane between the endothelium and the podocytes and the slit diaphragm between the interdigitating podocyte foot processes. All three parts are crucial for successful blood filtration. The elaborate glycocalyx covering the endothelial cell and its fenestrae already restricts particulate blood components and larger molecules like immunoglobulins and albumin (Schlöndorff 2014). The glomerular basement membrane and the slit diaphragm come into play as second and third level of filtration, all together providing the highly effective permselectivity of the filter and preventing it from clogging up (Schlöndorff 2014). A very elaborate and tightly regulated cross-talk between the glomerular cells is necessary for a fully functional renal filtration barrier.

5.1.1 Differences between animal model and cell culture

In metanephric organ culture, it has been shown that podocytes develop before the other cell types, hinting that an intrinsic development program is used for podocyte differentiation during development (Schlöndorff 2014). However, it is not known how a podocyte recognizes its neighboring podocyte to build up the interdigitating foot processes and the slit diaphragms in- between the processes. It has been shown that the fenestrae formation in the endothelium is caused by VEGF (Vascular endothelial growth factor) signaling, a signal protein produced by podocytes (Chen et al. 2002). The fenestration of the endothelium and its characteristic glycocalyx is dependent on podocyte VEGFA (Vascular endothelial growth factor A) and the presence of the fitting receptor, VEGFR-2 (Vascular endothelial growth factor receptor 2), in the endothelium (Schlöndorff 2014). While studies have elucidated the molecular basis of signaling from podocyte to endothelial cell and from endothelial cell to mesangial cell, potential mediators have yet to be identified for cross-talk between podocytes and mesangial cells as well as unidirectional signaling from endothelial cells to podocytes and from mesangial to endothelial cells (Schlöndorff 2014). A first hint for signaling from endothelial cells to the podocytes was

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given when medium conditioned from glomerular endothelial cells grown under permanent laminar shear stress decreased podocyte monolayer resistance and increased phosphorylation of vasodilator-stimulated phosphoprotein, showing a possible endothelium-to-podocyte crosstalk in cell culture (Slater et al. 2012).

The composition of the glomerular basement membrane has recently been studied by mass- spectrometry based proteomics (Byron et al. 2014). It was reported that cell type specific differences in the extra cellular matrix composition could be identified. Additionally, the extra cellular matrix deposition in co-cultured cells was different from the matrix in monocultures, hinting at cross-talk between the cell types. It has been tried to mimic the physiological state using hydrogel scaffolds (Bruggeman et al. 2012) or a bioartificial membranes containing collagen I (Slater et al. 2011) for culturing endothelial cells and podocytes on the two sides of a central substrate. But still, the very specialized three-dimensional podocyte architecture with the interdigitating foot processes of two neighboring podocytes embracing the capillaries has not been copied in cell culture yet.

Taken together, podocytes are placed in a very complex environment under physiological conditions, including a lot of signaling in-between the different cell types of the glomerulus. Additionally, the three-dimensional architecture of podocytes is not conserved in cells being cultured in two-dimensional cell culture. Thus, it is plausible that immortalized podocyte cell lines do not exhibit all characteristics of a podocyte in vivo.

5.1.2 Immortalized human cell line as model for podocytes

To investigate podocytes in cell culture, immortalized cell lines derived from isolated human and murine podocytes, that can be switched from a proliferating to a differentiated state, have been established (Saleem et al. 2002, Schiwek et al. 2004). For the human immortalized podocytes, the creators showed by immunofluorescence microscopy that the expression of nephrin as well as synaptopodin are low and diffuse in proliferating cells, but changes to a punctual pattern for nephrin and an actin associated pattern for synaptopodin in differentiated cells.

In the present work, the differentiation of immortalized human podocytes could also be nicely demonstrated by the changing localization of the podocyte marker nephrin, and the two actin associated adapter proteins synaptopodin and -actinin-4 between the proliferating and the differentiated state of hPCLs. Similar to the to the results published before (Saleem et al. 2002), it could be shown that nephrin relocalizes from a weak nuclear staining to a spot-like or linear localization at the cellular membrane in differentiated human podocytes. Synaptopodin localizes

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from nuclear staining with weak cytoplasmic staining to fibers spanning the whole differentiated cell, arguing for an actin colocalization of synaptopodin in differentiated hPCLs. Additionally, - actinin-4, an actin crosslinking protein known to be mutated in some patients with familial focal segmental glomerulosclerosis (Henderson et al. 2008), could be shown to relocate from fibers spanning the whole cytoplasm to “spike”-like protrusions at the cortex of the cells. All three proteins known to be important for podocyte structure show a distinct relocation pattern between the proliferating and the differentiated state, indicating a reorganization of the actin cytoskeleton and enhanced expression of nephrin and synaptopodin after differentiation. Additionally, the experiments showed that ten mature miRNAs that were found to be expressed in freshly isolated murine podocytes, miR-10b-5p, miR-92a-3p, miR-130a-3p, miR-30a-5p, miR- 146b-5p, miR-29a-3p, miR-23b-3p, miR-22-3p, miR-107 and miR-27b-3p, are also expressed in immortalized human podocytes. These miRNAs show differential expression between proliferating and differentiated hPCLs, also arguing for an altered metabolic and structural state of the differentiated cells. In contrast, the expression of another set of five mature miRNAs expressed in freshly isolated murine podocytes, miR-148a-3p, miR-196b-5p, miR-424-5p, miR- 542-3p and miR-503-5p, could neither be detected in the proliferating nor in the differentiated immortalized human podocytes. This shows either species specific expression of mature miRNAs or a gap between the situation of differentiated podocyte cell line and the physiological in vivo state. Since the immortalized murine podocytes can also be switched to the differentiated state by culturing them at 38°C for 14 days, expression of the miRNAs detected in freshly isolated murine podocytes might be checked in proliferating and differentiated mPCLs. Thus, it could be investigated if the differences are due to the different species or due to the difference between immortalized cell lines and in vivo podocytes.

The immortalized human podocytes show distinct differences between the proliferating and the differentiated state, in the expression and distribution of important structural proteins as well as their miRNA expression. Thus, they are a suitable model for regulatory mechanisms in human podocytes. There is, however, still a big difference between differentiated podocytes in the two- dimensional cell culture flask and the in vivo environment of podocytes. As freshly isolated human podocytes cannot be obtained, the immortalized human podocyte cell line is the best human model for experiments on the function of podocytes. For studies focusing on the delicate structure of podocytes in the mammalian organism, freshly isolated murine podocytes represent the most accurate model for podocyte structure and function under physiological conditions, since they are fully differentiated cells isolated from their natural environment.

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5.1.3 Freshly isolated murine podocytes

The major limitation in working with podocytes freshly isolated from reporter mice is the yield. By modifications of the original podocyte isolation protocol (Boerries et al. 2013), adding an additional rinsing step prior to perfusion of the kidney and prolonging the podocyte digestion step by five minutes, it was possible to increase the cell yield to an average of around 200,000 green fluorescent podocytes per animal in this work. This makes the isolation of five to six million cells a possible, but laborious task demanding many laboratory animals. Immortalized podocyte cell lines like the hPCLs and mPCLs can be expanded to millions of cells quite easily in a relatively short amount of time, while breading of a sufficient amount of laboratory animals takes several months and is considerably costlier.

The number of cells per glomerulus was estimated to be 211 ± 29 by the dissector/fractionator method (Basgen et al. 2006), while the number of podocytes per glomerulus was estimated to be around 89 ± 10 with the fractionator/dissector method (Nicholas et al. 2011). The number of nephrons in a healthy murine kidney has been investigated by several studies. By physical dissector/fractionator combination, the number of nephrons per kidney in 129Sv/C57bl/6 hybrid mice after the sixth backcross to C57bl/6 background was estimated to be 13440 ± 1275 (Cullen- McEwen et al. 2003). In a study focusing on the nephron number during kidney development, the number of nephrons in the adult kidney of four week old C57bl/6J mice varied between 9266 and 13453 with an mean value of 11359 when analyzed with the fractionator/dissector method (Zhong et al. 2012). Differences of nephron numbers between male and female mice have been reported, when 19341 ± 859 nephrons in male C57bl/6 mice, but 22830 ± 1117 nephrons in female C57bl/6 mice were counted in individuals of eight weeks of age (Murawski et al. 2010). By magnetic beads perfusion, 20,131 ± 4699 glomeruli could be isolated from mice with different backgrounds (C57bl/6, 129/SV or hybrids of the two strains, Takemoto 2002). Finally, it was reported that up to 500,000 podocytes can be isolated from one double fluorescent Cre reporter mouse (Boerries et al. 2013).

These numbers show that the number of nephrons, and thus, the number of podocytes per mouse varies with the age, sex, and genetic background of the laboratory animal, making an average of 200,000 podocytes per mouse a realistic yield.

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