Glomerular Endothelial Cell-Derived miR-200c Impairs Glomerular Homeostasis by Targeting Podocyte VEGF-A.

Deciphering the pathophysiological mechanisms of primary podocytopathies that can lead to end-stage renal disease and increased mortality is an unmet need. Studying how microRNAs (miRs) interfere with various signaling pathways enables identification of pathomechanisms, novel biomarkers and potential therapeutic options. We investigated the expression of miR-200c in urine from patients with different renal diseases as a potential candidate involved in podocytopathies. The role of miR-200c for the glomerulus and its potential targets were studied in cultured human podocytes, human glomerular endothelial cells and in the zebrafish model. miR-200c was upregulated in urine from patients with minimal change disease, membranous glomerulonephritis and focal segmental glomerulosclerosis and also in transforming growth factor beta (TGF-ß) stressed glomerular endothelial cells, but not in podocytes. In zebrafish, miR-200c overexpression caused proteinuria, edema, podocyte foot process effacement and glomerular endotheliosis. Although zinc finger E-Box binding homeobox 1/2 (ZEB1/2), important in epithelial to mesenchymal transition (EMT), are prominent targets of miR-200c, their downregulation did not explain our zebrafish phenotype. We detected decreased vegfaa/bb in zebrafish overexpressing miR-200c and could further prove that miR-200c decreased VEGF-A expression and secretion in cultured human podocytes. We hypothesize that miR-200c is released from glomerular endothelial cells during cell stress and acts in a paracrine, autocrine, as well as context-dependent manner in the glomerulus. MiR-200c can cause glomerular damage most likely due to the reduction of podocyte VEGF-A. In contrast, miR-200c might also influence ZEB expression and therefore EMT, which might be important in other conditions. Therefore, we propose that miR-200c-mediated effects in the glomerulus are context-sensitive.

A Tight Control of Non-Canonical TGF-ß Pathways and MicroRNAs Downregulates Nephronectin in Podocytes.

Nephronectin (NPNT) is an extracellular matrix protein in the glomerular basement membrane that is produced by podocytes and is important for the integrity of the glomerular filtration barrier. Upregulated transforming growth factor ß (TGF-ß) and altered NPNT are seen in different glomerular diseases. TGF-ß downregulates NPNT and upregulates NPNT-targeting microRNAs (miRs). However, the pathways involved were previously unknown. By using selective inhibitors of the canonical, SMAD-dependent, and non-canonical TGF-ß pathways, we investigated NPNT transcription, translation, secretion, and regulation through miRs in podocytes. TGF-ß decreased NPNT mRNA and protein in cultured human podocytes. TGF-ß-dependent regulation of NPNT was meditated through intracellular signaling pathways. Under baseline conditions, non-canonical pathways predominantly regulated NPNT post-transcriptionally. Podocyte NPNT secretion, however, was not dependent on canonical or non-canonical TGF-ß pathways. The canonical TGF-ß pathway was also dispensable for NPNT regulation after TGF-ß stimulation, as TGF-ß was still able to downregulate NPNT in the presence of SMAD inhibitors. In contrast, in the presence of different non-canonical pathway inhibitors, TGF-ß stimulation did not further decrease NPNT expression. Moreover, distinct non-canonical TGF-ß pathways mediated TGF-ß-induced upregulation of NPNT-targeting miR-378a-3p. Thus, we conclude that post-transcriptional fine-tuning of NPNT expression in podocytes is mediated predominantly through non-canonical TGF-ß pathways.

Glomerular Endothelial Cell-Derived microRNA-192 Regulates Nephronectin Expression in Idiopathic Membranous Glomerulonephritis.

BACKGROUND: Autoantibodies binding to podocyte antigens cause idiopathic membranous glomerulonephritis (iMGN). However, it remains elusive how autoantibodies reach the subepithelial space because the glomerular filtration barrier (GFB) is size selective and almost impermeable for antibodies. METHODS: Kidney biopsies from patients with iMGN, cell culture, zebrafish, and mouse models were used to investigate the role of nephronectin (NPNT) regulating microRNAs (miRs) for the GFB. RESULTS: Glomerular endothelial cell (GEC)-derived miR-192-5p and podocyte-derived miR-378a-3p are upregulated in urine and glomeruli of patients with iMGN, whereas glomerular NPNT is reduced. Overexpression of miR-192-5p and morpholino-mediated npnt knockdown induced edema, proteinuria, and podocyte effacement similar to podocyte-derived miR-378a-3p in zebrafish. Structural changes of the glomerular basement membrane (GBM) with increased lucidity, splitting, and lamellation, especially of the lamina rara interna, similar to ultrastructural findings seen in advanced stages of iMGN, were found. IgG-size nanoparticles accumulated in lucidity areas of the lamina rara interna and lamina densa of the GBM in npnt-knockdown zebrafish models. Loss of slit diaphragm proteins and severe structural impairment of the GBM were further confirmed in podocyte-specific Npnt knockout mice. GECs downregulate podocyte NPNT by transfer of miR-192-5p-containing exosomes in a paracrine manner. CONCLUSIONS: Podocyte NPNT is important for proper glomerular filter function and GBM structure and is regulated by GEC-derived miR-192-5p and podocyte-derived miR-378a-3p. We hypothesize that loss of NPNT in the GBM is an important part of the initial pathophysiology of iMGN and enables autoantigenicity of podocyte antigens and subepithelial immune complex deposition in iMGN.

Analysis of tetrodotoxin-sensitive sodium and low voltage-activated calcium channels in developing mouse retinal horizontal cells.

Expression patterns of voltage-gated ion channels determine the spatio-temporal dynamics of ion currents that supply excitable neurons in developing tissue with proper electrophysiological properties. The purpose of the study was to identify fast cationic inward currents in mouse retinal horizontal cells (HCs) and describe their biophysical properties at different developmental stages. We also aimed to reveal their physiological role in shaping light responses (LRs) in adult HCs. HCs were recorded in horizontal slices of wild-type mouse retina at postnatal stages ranging from p8 through p60. Voltage-dependent inward currents were isolated with appropriate voltage protocols and blockers specific for sodium and T-type calcium channels. LRs were evoked with full-field flashes (130 µW/cm2). Transient and steady inward currents were identified at all developmental stages. Transient currents were mediated by T-type calcium and TTX-sensitive sodium channels, whereas steady currents were blocked by cadmium, indicating the presence of high voltage-activated calcium channels. Activation and steady-state inactivation kinetics of T-type calcium channels revealed a contribution to the resting membrane potential during postnatal development. Additionally, both sodium and T-type calcium channels had an impact on HC LRs at light offset in adult animals. Our results showed that the voltage-dependent inward currents of postnatally developing mouse HCs consist of T-type calcium, TTX-sensitive sodium, and high voltage-activated calcium channels, and that transient ionic currents contributed to light-evoked responses of adult HCs, suggesting a role in HC information processing.

A network of trans-cortical capillaries as mainstay for blood circulation in long bones.

Closed circulatory systems (CCS) underlie the function of vertebrate organs, but in long bones their structure is unclear, although they constitute the exit route for bone marrow (BM) leukocytes. To understand neutrophil emigration from BM, we studied the vascular system of murine long bones. Here we show that hundreds of capillaries originate in BM, cross murine cortical bone perpendicularly along the shaft and connect to the periosteal circulation. Structures similar to these trans-cortical-vessels (TCVs) also exist in human limb bones. TCVs express arterial or venous markers and transport neutrophils. Furthermore, over 80% arterial and 59% venous blood passes through TCVs. Genetic and drug-mediated modulation of osteoclast count and activity leads to substantial changes in TCV numbers. In a murine model of chronic arthritic bone inflammation, new TCVs develop within weeks. Our data indicate that TCVs are a central component of the CCS in long bones and may represent an important route for immune cell export from the BM.