Spatiotemporal organisation of protein processing in the kidney

The kidney regulates plasma protein levels by eliminating them from the circulation. Proteins filtered by glomeruli are endocytosed and degraded in the proximal tubule and defects in this process result in tubular proteinuria, an important clinical biomarker. However, the spatiotemporal organization of renal protein metabolism in vivo was previously unclear. Here, using functional probes and intravital microscopy, we track the fate of filtered proteins in real time in living mice, and map specialized processing to tubular structures with singular value decomposition analysis and three-dimensional electron microscopy. We reveal that degradation of proteins requires sequential, coordinated activity of distinct tubular sub-segments, each adapted to specific tasks. Moreover, we leverage this approach to pinpoint the nature of endo-lysosomal disorders in disease models, and show that compensatory uptake in later regions of the proximal tubule limits urinary protein loss. This means that measurement of proteinuria likely underestimates severity of endocytotic defects in patients

Multisystem involvement, defective lysosomes, and impaired autophagy in a novel rat model of Nephropathic Cystinosis

Recessive mutations in the CTNS gene encoding the lysosomal transporter cystinosin cause cystinosis, a lysosomal storage disease leading to kidney failure and multisystem manifestations. A Ctns knock-out mouse model recapitulates features of cystinosis, but the delayed onset of kidney manifestations, phenotype variability, and strain effects limit its use for mechanistic and drug development studies. To provide a better model for cystinosis, we generated a Ctns knock-out rat model using CRISPR/Cas9 technology. The Ctns-/- rats display progressive cystine accumulation and crystal formation in multiple tissues including kidney, liver and thyroid. They show an early onset and progressive loss of urinary solutes, indicating generalized proximal tubule dysfunction, with development of typical swan-neck lesions, tubulointerstitial fibrosis and kidney failure, and decreased survival. The Ctns-/- rats also present crystals in the cornea, and bone and liver defects, like in patients. Mechanistically, the loss of cystinosin induces a phenotype switch associating abnormal proliferation and dedifferentiation, loss of apical receptors and transporters, and defective lysosomal activity and autophagy in the cells. Primary cultures of proximal tubule cells derived from the Ctns-/- rat kidneys confirmed the key changes caused by cystine overload, including reduced endocytic uptake, increased proliferation and defective lysosomal dynamics and autophagy. The novel Ctns-/- rat model and derived proximal tubule cell system provide invaluable tools to investigate the pathogenesis of cystinosis and to accelerate drug discovery

The proximal tubule, protein uptake, and the riddle of the segments

The proximal tubule is divided anatomically into 3 distinct segments-S1 to S3-on the basis of differences in cellular ultrastructure, but the functional processes that define and shape these remain elusive. In a new study, Christensen used 3-dimensional nephron reconstruction, electron microscopy, and antibody staining to precisely map protein uptake to the structure of the proximal tubule. They reported striking axial patterns in endocytosis along the segments, which showed substantial plasticity in disease states

Implementation of space charge measurement using the Pulsed Electro- Acoustic method during ageing of HVDC model cable

International audienceDue to high dielectric performances, cross-linked polyethylene (XLPE) is one of the preferred materials for the insulation of HVDC cables. However, polymeric insulating materials tend to store electric charges, which disturb the electric field within the material and may eventually lead to a structural modification of the polymer thus promoting its ageing. This degradation of the insulation may induce a decrease in the life expectancy of the cables. Nowadays many studies are carried out to bring knowledge on the degradation mechanisms in polyethylene. Indeed, an improved understanding of the behaviour of charges makes it possible to better correlate the measurements with the ageing of the material. However, it is often not possible to measure the dielectric characteristics (current, space charge) during ageing, and most generally this has to be done a posteriori. The objective of this research is to measure the variations of the dielectric properties of the polyethylene insulation in a model cable, either during ageing conditions, i.e. when a thermo-electric stress is applied to the cable, or a posteriori. To do so, two approaches have been developed and put into practice. The first approach is based on the adaptation of the Pulsed Electro-Acoustic (PEA) measurement method to an already charged model cable having undergone thermo-electric ageing. In this case, the PEA measurement is not performed online, but on samples taken regularly from a long cable length under thermo-electric stress. Hence, for each measurement, a different piece of cable, already charged when received, is under study. The PEA method requires a calibration step, normally performed on a non-charged sample to obtain the response of the system. We have developed a calibration procedure on an already charged cable without removing the possible charges that are present in the insulation. In this way crucial information on the state of charge before any further electrical stressing can be analysed. The treatment of the PEA signal has been revised, and it is now possible to quantify the charge present on an already stressed cable sample prior to any measurement. The improvement of the PEA signal conditioning will be presented in the paper. Moreover, the results of space charge measurements at different ageing times will be discussed

Effect of Gamma Irradiation Dose on Space Charge in e-Beam Irradiated PET Films

International audienceInvestigating radiation-induced effects in synthetic polymers makes sense for nuclear power and aerospace industries. We propose in this work to investigate the effect of the dose pre-deposited by gamma rays on the behavior of the charges implanted by electron beam in PET (polyethylene terephthalate) samples. The samples used in this study are PET films of 25 µm in thickness. The samples were first irradiated by gamma rays, at different doses (0.1 to 2 MGy). Then, they were exposed to 20 keV energy electron beam, during 5 min, with the purpose to implant electrons near the surface. Materials were characterized by different techniques, notably Light Intensity Modulation Method (LIMM) for space charge measurements and FTIR as physicochemical analysis. The results show that the electrons implanted in the PET irradiated at high dose do not stay close to the surface for a long time, which could be associated with significant material modifications at the scale of the molecules

Multisystem involvement, defective lysosomes and impaired autophagy in a novel rat model of nephropathic cystinosis

Recessive mutations in the CTNS gene encoding the lysosomal transporter cystinosin cause cystinosis, a lysosomal storage disease leading to kidney failure and multisystem manifestations. A Ctns knockout mouse model recapitulates features of cystinosis, but the delayed onset of kidney manifestations, phenotype variability and strain effects limit its use for mechanistic and drug development studies. To provide a better model for cystinosis, we generated a Ctns knockout rat model using CRISPR/Cas9 technology. The Ctns(−/−) rats display progressive cystine accumulation and crystal formation in multiple tissues including kidney, liver and thyroid. They show an early onset and progressive loss of urinary solutes, indicating generalized proximal tubule dysfunction, with development of typical swan-neck lesions, tubulointerstitial fibrosis and kidney failure, and decreased survival. The Ctns(−/−) rats also present crystals in the cornea, and bone and liver defects, as observed in patients. Mechanistically, the loss of cystinosin induces a phenotype switch associating abnormal proliferation and dedifferentiation, loss of apical receptors and transporters, and defective lysosomal activity and autophagy in the cells. Primary cultures of proximal tubule cells derived from the Ctns(−/−) rat kidneys confirmed the key changes caused by cystine overload, including reduced endocytic uptake, increased proliferation and defective lysosomal dynamics and autophagy. The novel Ctns(−/−) rat model and derived proximal tubule cell system provide invaluable tools to investigate the pathogenesis of cystinosis and to accelerate drug discovery

Impaired mitophagy links mitochondrial disease to epithelial stress in methylmalonyl-CoA mutase deficiency

Deregulation of mitochondrial network in terminally differentiated cells contributes to a broad spectrum of disorders. Methylmalonic acidemia (MMA) is one of the most common inherited metabolic disorders, due to deficiency of the mitochondrial methylmalonyl-coenzyme A mutase (MMUT). How MMUT deficiency triggers cell damage remains unknown, preventing the development of disease–modifying therapies. Here we combine genetic and pharmacological approaches to demonstrate that MMUT deficiency induces metabolic and mitochondrial alterations that are exacerbated by anomalies in PINK1/Parkin–mediated mitophagy, causing the accumulation of dysfunctional mitochondria that trigger epithelial stress and ultimately cell damage. Using drug–disease network perturbation modelling, we predict targetable pathways, whose modulation repairs mitochondrial dysfunctions in patient–derived cells and alleviate phenotype changes in mmut–deficient zebrafish. These results suggest a link between primary MMUT deficiency, diseased mitochondria, mitophagy dysfunction and epithelial stress, and provide potential therapeutic perspectives for MMA

The Standard.

Fundación Bunge y Born.AECID