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Background Renal ischemia-reperfusion (I/R) injury is a major cause of AKI. Noncoding RNAs are intricately involved in the pathophysiology of this form of AKI. Transcription of hypoxia-induced, long noncoding RNA H19, which shows high embryonic expression and is silenced in adults, is upregulated in renal I/R injury. Methods Lentivirus-mediated overexpression, as well as antisense oligonucleotide-based silencing, modulated H19 in vitro. In vivo analyses used constitutive H19 knockout mice. In addition, renal vein injection of adeno-associated virus 2 (AAV2) carrying H19 caused overexpression in the kidney. Expression of H19 in kidney transplant patients with I/R injury was investigated. Results H19 is upregulated in kidney biopsies of patients with AKI, in murine ischemic kidney tissue, and in cultured and ex vivo sorted hypoxic endothelial cells (ECs) and tubular epithelial cells (TECs). Transcription factors hypoxia-inducible factor 1-α, LHX8, and SPI1 activate H19 in ECs and TECs. H19 overexpression promotes angiogenesis in vitro and in vivo. In vivo, transient AAV2-mediated H19 overexpression significantly improved kidney function, reduced apoptosis, and reduced inflammation, as well as preserving capillary density and tubular epithelial integrity. Sponging of miR-30a-5p mediated the effects, which, in turn, led to target regulation of Dll4, ATG5, and Snai1. Conclusions H19 overexpression confers protection against renal injury by stimulating proangiogenic signaling. H19 overexpression may be a promising future therapeutic option in the treatment of patients with ischemic AKI.

BACKGROUND Circular RNAs (circRNAs) have recently been described as novel noncoding regulators of gene expression. They are detectable in the blood of patients with acute kidney injury. We tested whether circRNAs were present in urine and could serve as new predictors of outcome in renal transplant patients with acute rejection. METHODS A global circRNA expression analysis using RNA from urine of patients with acute T cell-mediated renal allograft rejection and control transplant patients was performed. Dysregulated circRNAs were confirmed in a cohort of 62 patients with acute rejection, 10 patients after successful antirejection therapy, 18 control transplant patients without rejection, and 13 stable transplant patients with urinary tract infection. RESULTS A global screen revealed several circRNAs to be altered in urine of patients with acute rejection. Concentrations of 2 circRNAs including hsa_circ_0001334 and hsa_circ_0071475 were significantly increased. These were validated in the whole cohort of patients. hsa_circ_0001334 was upregulated in patients with acute rejection compared with controls. Concentrations of hsa_circ_0001334 normalized in patients with acute rejection following successful antirejection therapy. hsa_circ_0001334 was associated with higher decline in glomerular filtration rate 1 year after transplantation. CONCLUSIONS CircRNA concentrations are significantly dysregulated in patients with acute rejection at subclinical time points. Urinary hsa_circ_0001334 is a novel biomarker of acute kidney rejection, identifying patients with acute rejection and predicting loss of kidney function.

Primary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1-positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induced proteolysis of the 2 essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1 IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1 titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 were required to induce proteolysis of synaptopodin and NEPH1 by 2 distinct proteolytic pathways mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrated a mechanism by which aberrantly glycosylated IgG4 activated the lectin pathway and induced podocyte injury in primary membranous nephropathy.

Secondary Ion Mass Spectrometry (SIMS) is a well-established and extremely powerful technique for the chemical analysis of surfaces and thin films. Its main advantages are its excellent sensitivity, its high dynamic range, its good mass resolution and its ability to distinguish between isotopes. Due to its excellent sensitivity and thus its low detection limits, SIMS can be used to detect both major and trace elements. While SIMS was originally mainly used for depth profiling, the applications gradually shifted towards 2D and 3D imaging as a result of the dramatically improved spatial resolution resulting from the progress made on the instrumental side. As a consequence, new fields of application for SIMS, e.g. in life sciences and nanotechnologies, are emerging. In addition, the possibility of detecting several isotopes in parallel opens still other horizons, mainly in life sciences, where isotopic labeling is an important investigation technique. Traditional SIMS 3D imaging is however affected by serious artifacts: while these traditional 3D reconstruction protocols and software assume that the initial sample surface is flat and the analyzed volume is cuboid, “real samples” present a surface topography, which furthermore changes during the ion bombardment as the local sputter yields depend on parameters such as the local angle of incidence of the ion beam and the crystal orientation. In addition, the situation is worsened if the sample is constituted of different materials due to preferential sputtering phenomena. As a consequence, the produced 3D images are affected by a more or less important uncertainty on the depth scale and can be distorted. Finally, significant field inhomogeneities arise from the surface topography as a result of distortion of the local electric field. These perturb both the primary beam and the trajectories of secondary ions, resulting in a number of possible artifacts, including shifts in apparent pixel position and changes in intensity. Attempts to overcome this limitation have been done by performing ex-situ Atomic Force Microscopy (AFM) measurements on the sample surface before the SIMS analysis and on the post bombardment craters. However, the accuracy of this approach is limited. At first, the obtained AFM images only reflect the situation before or after bombardment, but give no intermediate information regarding the evolution of the roughness during sputtering. Secondly, the fact that the sample needs to be exposed to air while transferring it from the SIMS instrument to the AFM has shown to introduce considerable artifacts due to surface oxidation and surface reorganization. This also applies to samples previously analyzed with a Cs + primary ion beam, which is the case on many state-of-the-art SIMS instruments, as air exposure leads to the formation of so-called Cs dots on the sample, which again then considerably changes the surface topography of the sample. Thirdly, as (organic) SIMS measurements are frequently performed at a controlled low temperature of the sample, ex-situ AFM analysis will lead to artifacts, as the ambient conditions are different between SIMS and AFM.

With the breakthroughs in lateral resolution with regards to secondary ion mass spectroscopy in recent years, new areas of research with much promise have opened up to the scientific community. Even though the much improved lateral resolution of 50 nm can effectively deliver more accurate 3D-images, the traditional 3D reconstructions, consisting of compiling previously acquired successive secondary ion mass spectrometry images into a 3D-stack, do not represent the real localized chemical distribution of the sputtered volume. Based on samples initially analyzed on the Cameca NanoSIMS 50 instrument, this paper portrays the advantages of combining the topographical information from atomic force microscopy and the chemical information from secondary ion mass spectrometry. Taking account of the roughness evolution within the analyzed zone, 3D reconstructions become a lot more accurate and allow an easier interpretation of results. On the basis of an Al/Cu sample, a comparison between traditional 3D imaging and corrected 3D reconstructions is given and the advantages of the newly developed 3D imaging method are explained.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

The purpose of this research was to study the validity and variability of a projection Moire system, measuring volume differences of geometrically different formed specimens mimicking localized alveolar ridge defects. Nine pairs of specimens were fabricated, each of which simulated a preoperative ridge defect and a corresponding surgically-corrected postoperative ridge defect. All specimen pairs had a mathematically defined form which allowed the accurate assessment of their volume differences by a mechanical 3-D coordinate measuring machine or by a software-controlled milling machine. Measurements achieved with these methods were used as the references for comparison. Six specimen pairs, A1 to A6, possessed a simple rectangular geometrical form which facilitated their fabrication. Three specimen pairs, B1 to B3, were milled and consisted of geometrically more complex 3-D sculptured surfaces, which came closest to a true imitation of a localized ridge defect. An optical measurement system in the form of the projection Moire was utilized, applying a 4-phase shift technique, and results obtained with this device were regarded as test volumes. The absolute variability of the test volume measurements differed between 0.397 mm3 to 15.872 mm3, corresponding to a relative variability of 0.83% to 2.83%. The mean of the relative variability was within 1.68% for the "A" specimens and 2.15% for the "B" specimens. However, the difference was not significant, probably due to the limited number of "B" specimens. The systematic error of the Moire measurements in relation to the reference methods was surprisingly low, ranging from -0.12 mm3 to 7.67 mm3. The relative systematic error, expressed as a percentage of reference volume, ranged between 0.06% and -2.23%. The mean of the relative error for the more complex "B" specimens was 1.37%, which was less accurate in comparison to the more simply formed "A" specimens with a relative systematic error of 0.35%. Therefore, in this in vitro model it was possible to measure volume differences of geometrically different formed specimens, mimicking localized alveolar ridge defects, with a validity within 2.2% and with a variability of less than 2.8%.

State-of-the-art secondary ion mass spectrometry (SIMS) instruments allow producing 3D chemical mappings with excellent sensitivity and spatial resolution. Several important artifacts however arise from the fact that SIMS 3D mapping does not take into account the surface topography of the sample. In order to correct these artifacts, we have integrated a specially developed scanning probe microscopy (SPM) system into a commercial Cameca NanoSIMS 50 instrument. This new SPM module, which was designed as a DN200CF flange-mounted bolt-on accessory, includes a new high-precision sample stage, a scanner with a range of 100 μm in x and y direction, and a dedicated SPM head which can be operated in the atomic force microscopy (AFM) and Kelvin probe force microscopy modes. Topographical information gained from AFM measurements taken before, during, and after SIMS analysis as well as the SIMS data are automatically compiled into an accurate 3D reconstruction using the software program "SARINA," which was developed for this first combined SIMS-SPM instrument. The achievable lateral resolutions are 6 nm in the SPM mode and 45 nm in the SIMS mode. Elemental 3D images obtained with our integrated SIMS-SPM instrument on Al/Cu and polystyrene/poly(methyl methacrylate) samples demonstrate the advantages of the combined SIMS-SPM approach.