Podocytes Produce and Secrete Functional Complement C3 and Complement Factor H

Podocytes are an important part of the glomerular filtration barrier and the key player in the development of proteinuria, which is an early feature of complement mediated renal diseases. Complement factors are mainly liver-born and present in circulation. Nevertheless, there is a growing body of evidence for additional sites of complement protein synthesis, including various cell types in the kidney. We hypothesized that podocytes are able to produce complement components and contribute to the local balance of complement activation and regulation. To investigate the relevant balance between inhibiting and activating sides, our studies focused on complement factor H (CFH), an important complement regulator, and on C3, the early key component for complement activation. We characterized human cultured podocytes for the expression and secretion of activating and regulating complement factors, and analyzed the secretion pathway and functional activity. We studied glomerular CFH and C3 expression in puromycin aminonucleoside (PAN) -treated rats, a model for proteinuria, and the physiological mRNA-expression of both factors in murine kidneys. We found, that C3 and CFH were expressed in cultured podocytes and expression levels differed from those in cultivated glomerular endothelial cells. The process of secretion in podocytes was stimulated with interferon gamma and located in the Golgi apparatus. Cultured podocytes could initiate the complement cascade by the splitting of C3, which can be shown by the generation of C3a, a functional C3 split product. C3 contributed to external complement activation. Podocyte-secreted CFH, in conjunction with factor I, was able to split C3b. Podocytes derived from a patient with a CFH mutation displayed impaired cell surface complement regulation. CFH and C3 were synthesized in podocytes of healthy C57Bl/6-mice and were upregulated in podocytes of PAN treated rats. These data show that podocytes produce functionally active complement components, and could therefore influence the local glomerular complement activation and regulation. This modulating effect should therefore be considered in all diseases where glomerular complement activation occurs. Furthermore, our data indicate a potential novel role of podocytes in the innate immune system

Accumulation of α-synuclein mediates podocyte injury in Fabry nephropathy

Current therapies for Fabry disease are based on reversing intracellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosomal dysfunction. However, their effect in the reversal of end-organ damage, like kidney injury and chronic kidney disease, remains unclear. In this study, ultrastructural analysis of serial human kidney biopsies showed that long-term use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/Cas9–mediated α-galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified α-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.publishedVersio

Stacked and Tunable Large-Scale Plasmonic Nanoparticle Arrays for Surface-Enhanced Raman Spectroscopy

Surface-enhanced Raman spectroscopy takes advantage of plasmonic substrates that sustain resonances at tunable frequencies with a reproducibly extraordinary field enhancement. Low-cost and large-scale fabrication of these substrates is further required. Here, we present stacked large-scale arrays of strongly coupled gold nanoparticles as promising candidates for such substrates. These arrays are fabricated by bottom-up techniques that fulfill the aforementioned requirements. The distance between adjacent arrays in the stack is controlled with high precision using a discrete number of monolayers of molecules that enable the spectral position of the plasmonic resonances to be tuned. Although the nanoparticles are randomly arranged in each array, the spatial proximity of the stacked arrays enables a strong coupling among nanoparticles to be achieved in adjacent arrays. The huge field enhancements due to these strongly coupled gold nanoparticles are shown to enhance the Raman signal. We show that effectively the optical response from these stacked arrays and the Raman signals can be understood in a simplifying picture where only an individual nanoparticle dimer is considered. The possibility to tune the plasmonic resonances of the substrate across the visible spectrum makes our material a plasmonic substrate of choice for many applications where light–matter interactions need to be intensified

Plasma-Enhanced Atomic Layer Deposition of HfO2 with Substrate Biasing: Thin Films for High-Reflective Mirrors

Tuning ion energies in plasma-enhanced atomic layer deposition (PEALD) processes enables fine control over the material properties of functional coatings. The growth, structural, mechanical, and optical properties of HfO2 thin films are presented in detail toward photonic applications. The influence of the film thickness and bias value on the properties of HfO2 thin films deposited at 100 °C using tetrakis(dimethylamino)hafnium (TDMAH) and oxygen plasma using substrate biasing is systematically analyzed. The HfO2 films deposited without a substrate bias show an amorphous microstructure with a low density, low refractive index, high incorporation of residual hydroxyl (OH) content, and high residual tensile stress. The material properties of HfO2 films significantly improved at a low bias voltage due to the interaction with oxygen ions accelerated to the film. Such HfO2 films have a higher density, higher refractive index, and lower residual OH incorporation than films without bias. The mechanical stress becomes compressive depending on the bias values. Further increasing the ion energies by applying a larger substrate bias results in a decrease of the film density, refractive index, and a higher residual OH incorporation as well as crystalline inclusions. The comparable material properties of the HfO2 films have been reported using tris(dimethylamino)cyclopentadienyl hafnium (TDMACpH) in a different apparatus, indicating that this approach can be transferred to various systems and is highly versatile. Finally, the substrate biasing technique has been introduced to deposit stress-compensated, crack- and delamination-free high-reflective (HR) mirrors at 355 and 532 nm wavelengths using HfO2 and SiO2 as high and low refractive index materials, respectively. Such mirrors could not be obtained without the substrate biasing during the deposition because of the high tensile stress of HfO2, leading to cracks in thick multilayer systems. An HR mirror for 532 nm wavelength shows a high reflectance of 99.93%, a residual transmittance of ∼530 ppm, and a low absorption of ∼11 ppm, as well as low scattering losses of ∼4 ppm, high laser-induced damage threshold, low mechanical stress, and high environmental stability