Chronic kidney disease and diabetes mellitus are associated with extensive media calcification, which leads to increased cardiovascular morbidity and mortality. Here, we investigated the role of autophagy in the pathogenesis of uremic vascular media calcification. DBA/2 mice were fed with high-phosphate diet (HPD) in order to cause vascular calcification. DBA/2 mice on standard chow diet were used as control. In parallel, autophagy and its response to rapamycin, 3-methyladenine (3-MA), and bafilomycin were studied in an in vitro model using mouse vascular smooth muscle cells (MOVAS). DBA/2 mice on HPD developed severe vascular media calcification, which is mirrored in vitro by culturing MOVAS under calcifying conditions. Both, in vitro and in vivo, autophagy significantly increased in MOVAS under calcifying conditions and in aortas of HPD mice, respectively. Histologically, autophagy was located to the aortic Tunica media, but also vascular endothelial cells, and was found to continuously increase during HPD treatment. 3-MA as well as bafilomycin blocked autophagy in MOVAS and increased calcification. Vice versa, rapamycin treatment further increased autophagy and resulted in a significant decrease of vascular calcification in vitro and in vivo. Rapamycin reduced Runx2 transcription levels in aortas and MOVAS to control levels, whereas it increased α-smooth muscle actin and Sm22α transcription in MOVAS to control levels. Furthermore, rapamycin-treated HPD mice survived significantly longer compared to HPD controls. These findings indicate that autophagy is an endogenous response of vascular smooth muscle cells (VSMC) to protect from calcification in uremia. Induction of autophagy by rapamycin protects cells and mice from uremic media calcification possibly by inhibiting osteogenic transdifferentiation of VSMC.

Glucagon-like peptide (GLP)-1 analogs such as liraglutide improved albuminuria in patients with type 2 diabetes in large randomized controlled trials. One of the suspected mechanisms is the antiinflammatory potential of GLP-1 receptor (Glp1r) agonism. Thus, the anti-inflammatory action of Glp1r agonism was tested in a nondiabetic, T-cellemediated murine model of nephrotoxic serum nephritis (NTS). The role of Glp1r in NTS was evaluated by using Glp1r À/À mice or C57BL/6 mice treated with liraglutide. In vitro, murine T cells were stimulated in the presence of liraglutide or vehicle. Glp1r À/À mice displayed increased renal infiltration of neutrophils and T cells after induction of NTS. Splenocyte proliferation and TH1 cytokine transcription were increased in spleen and lymph nodes of Glp1r À/À mice. Liraglutide treatment significantly improved the renal outcome of NTS in C57BL/6 mice by decreasing renal infiltration and proliferation of T cells, which resulted in decreased macrophage infiltration. In vitro, T cells stimulated in the presence of liraglutide showed decreased proliferation of TH1 and TH17 cells. Liraglutide blocked glycolysis in T cells and decreased their Glut1 mRNA expression. Together, Glp1r agonism protects mice from a T-celledependent glomerulonephritis model by inhibition of T-cell proliferation, possibly by interacting with their metabolic program. This mechanism may explain in part the renoprotective effects of Glp1r agonism in diabetic nephropathy.

Background and aims Chronic kidney disease (CKD) is strongly associated with a high burden of cardiovascular morbidity and mortality. Therefore, we aimed to characterize the putative role of microRNAs (miR)s in uremic vascular remodelling and endothelial dysfunction. Methods We investigated the expression pattern of miRs in two independent end-stage renal disease (ESRD) cohorts and in the animal model of uremic DBA/2 mice via quantitative RT-PCR. Moreover, DBA/2 mice were treated with intravenous injections of synthetic miR-142-3p mimic and were analysed for functional and morphological vascular changes by mass spectrometry and wire myography. Results The expression pattern of miRs was regulated in ESRD patients and was reversible after kidney transplantation. Out of tested miRs, only blood miR-142-3p was negatively associated with carotid-femoral pulse-wave velocity in CKD 5D patients. We validated these findings in a murine uremic model and found similar suppression of miR-142-3p as well as decreased acetylcholine-mediated vascular relaxation of the aorta. Therefore, we designed experiments to restore bioavailability of aortic miR-142-3p in vivo via intravenous injection of synthetic miR-142-3p mimic. This intervention restored acetylcholine-mediated vascular relaxation. Conclusions Taken together, we provide compelling evidence, both in humans and in mice, that miR-142-3p constitutes a potential pharmacological agent to prevent endothelial dysfunction and increased arterial stiffness in ESRD.

Chronic kidney disease (CKD) is associated with mineral and bone disorder (MBD), which is the main cause of the extensively increased cardiovascular mortality in the CKD population. We now aimed to establish a new murine experimental CKD-MBD model. Dilute brown non-Agouti (DBA/2) mice were fed with high-phosphate diet for 4 (HPD4) or 7 (HPD7) days, then with standard chow diet (SCD) and subsequently followed until day 84. They were compared to DBA/2 mice maintained on SCD during the whole study period. Both 4 and 7 days HPD-fed mice developed phosphate nephropathy with tubular atrophy, interstitial fibrosis, decreased glomerular filtration rate, and increased serum urea levels. The abdominal aorta of HPD-treated mice showed signs of media calcification. Histomorphometric analysis of HPD-treated mice showed decreased bone volume/tissue volume, low mineral apposition rate, and low bone formation rate as compared to SCD-fed mice, despite increased parathyroid hormone levels. Overall, the observed phenotype was more pronounced in the HPD7 group. In summary, we established a new, noninvasive, and therefore easy to perform reproducible CKD-MBD model, which showed media calcification, secondary hyperparathyroidism, and low-turnover bone disease.

Background and Aims Podocin is a major component of the glomerular slit diaphragm. It is encoded by NPHS2, the most frequently mutated gene in steroid-resistant nephrotic syndrome. We previously showed that podocin oligomerization occurs exclusively through the C-terminal helical region and is responsible for mediating interallelic interactions of NPHS2. Podocin anchors the main slit diaphragm component nephrin to the podocyte cell membrane. The shortest dimension of the glomerular pore (3,5-4nm) is determined by the distance between two neighboring nephrin molecules in cis position and – according to our structural model – corresponds perfectly to the distance between the nephrin binding domains of two podocin molecules in an oligomer. We therefore hypothesized that podocin regulates the distance between nephrin molecules, thus affecting the size of the glomerular pore. Here we aimed to explore the effects of podocin variants on the distance between neighboring nephrin molecules. Method Wild type nephrin was tagged with C-terminal YPet or mRuby3 fluorescent proteins and the two constructs were transiently coexpressed in HEK293 cells, together with podocin variants of different oligomer-forming capacity. We verified the plasmamembrane localization of the fluorescently labelled nephrin molecules by confocal microscopy. Förster resonance energy transfer (FRET) was measured between the fluorescently labelled nephrin molecules in living cells. In a second set of experiments the fluorescent tags (Ypet and mCherry) were inserted in the extracellular part of nephrin at a distance of ten amino acids from the N-terminal end of the transmembrane domain. The fluorescence decay curves measured by time-correlated single photon counting (TCSPC) (Chronos BH, ISS Inc.) were decomposed into lifetime components, and the longest lifetime population – most suitable for long-range FRET – was used for further calculations. Nonparametric tests were used to analyze the data. Results Similar results were obtained with the extracellular and the intracellular tags. FRET efficiency was increased in the presence of the wild type podocin (p<0.0005 with both extracellular and intracellular tags), but remained unaltered in the presence of the monomer-forming R286Tfs*17 podocin. Similar to R286Tfs*17, the other pathogenic podocin variants (R138Q, A284V and F344Lfs*4) did not alter the FRET efficiency, irrespective of the different effect of these mutations on podocin oligomerization and localization. On the other hand, the coexpression of the R229Q podocin variant, which is benign in the homozygous state, resulted in a similarly high FRET efficiency as the wild type podocin. The FRET efficiency measured between extracellularly labelled nephrin molecules in the presence of the V290M podocin variant correlated with its clinically hypomorphic effect: it fell between that of the wild type and the R286Tfs*17 podocin, being statistically different from both. Conclusion Wild type podocin significantly reduces the distance between the nephrin molecules in cis: the shortest dimension of the glomerular pore. Several pathogenic podocin variants do not. The FRET efficiency between the nephrin molecules correlates with the clinical effect of the podocin variants. We thus suggest that a major function of podocin is the regulation of the critical dimension of the glomerular pore.