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Reactive carbonyl species (RCS) such as methylglyoxal (MGO) and glyoxal (GO) are highly reactive, unwanted side-products of cellular metabolism maintained at harmless intracellular levels by specific scavenging mechanisms.MGO and GO are metabolized through the glyoxalase (GLX) system, which consists of two enzymes acting in sequence, GLXI and GLXII. While plant genomes encode a number of different GLX isoforms, their specific functions and how they arose during evolution are unclear. Here, we used Arabidopsis (Arabidopsis thaliana) as a model species to investigate the evolutionary history of GLXI and GLXII in plants and whether the GLX system can protect plant cells from the toxicity of RCS other than MGO and GO. We show that plants possess two GLX systems of different evolutionary origins and with distinct structural and functional properties. The first system is shared by all eukaryotes, scavenges MGO and GO, especially during seedling establishment, and features Zn2+-type GLXI proteins with a metal cofactor preference that were present in the last eukaryotic common ancestor. GLXI and GLXII of the second system, featuring Ni2+-type GLXI, were acquired by the last common ancestor of Viridiplantae through horizontal gene transfer from proteobacteria and can together metabolize keto-D-glucose (KDG, glucosone), a glucose-derived RCS, to D-gluconate. When plants displaying loss-of-function of a Viridiplantae-specific GLXI were grown in KDG, D-gluconate levels were reduced to 10%–15% of those in the wild type, while KDG levels showed an increase of 48%–67%. In contrast to bacterial GLXI homologs, which are active as dimers, plant Ni2+-type GLXI proteins contain a domain duplication, are active as monomers, and have a modified second active site. The acquisition and neofunctionalization of a structurally, biochemically, and functionally distinct GLX system indicates that Viridiplantae are under strong selection to detoxify diverse RCS.

A high prevalence of AA-amyloidosis was identified in a breeding colony of northern tree shrews ( Tupaia belangeri) in a retrospective analysis, with amyloid deposits in different organs being found in 26/36 individuals (72%). Amyloid deposits, confirmed by Congo red staining, were detected in kidneys, intestines, skin, and lymph nodes, characteristic of systemic amyloidosis. Immunohistochemically, the deposited amyloid was intensely positive with anti-AA-antibody (clone mc4), suggesting AA-amyloidosis. The kidneys were predominantly affected (80%), where amyloid deposits ranged from mild to severe and was predominantly located in the renal medulla. In addition, many kidneys contained numerous cysts with atrophy of the renal parenchyma. There was no significant association between concurrent neoplastic or inflammatory processes and amyloidosis. The lack of distinctive predisposing factors suggests a general susceptibility of captive T. belangeri to develop amyloidosis. Clinical and laboratory findings of a female individual with pronounced kidney alterations were indicative of renal failure. The observed tissue tropism with pronounced kidney alterations, corresponding renal dysfunction, and an overall high prevalence suggests amyloidosis as an important disease in captive tree shrews.

Reactive carbonyl species (RCS) such as methylglyoxal (MGO) and glyoxal (GO) are highly reactive, unwanted side-products of cellular metabolism maintained at harmless intracellular levels by specific scavenging mechanisms. MGO and GO are metabolized through the glyoxalase (GLX) system, which consists of two enzymes acting in sequence, GLXI and GLXII. While plant genomes encode a large number of different GLX isoforms, it is unclear what their specific functions are and how these arose in evolution. Here, we show that plants possess two GLX systems of different evolutionary origins and with distinct structural and functional properties. The first system is shared by all eukaryotes, scavenges MGO and GO especially during seedling establishment, and features Zn2+-type GLXI, a metal co-factor preference that arose already in the last eukaryotic common ancestor. The GLXI and GLXII of the second system can together metabolize KDG, a glucose-derived RCS, and were acquired by the last common ancestor of viridiplantae through horizontal gene transfer from proteobacteria. In contrast to bacterial GLXI homologs, which are active as dimers, plant Ni2+-type GLXI contain a domain duplication, are active as monomers, and have modified their second active site. The acquisition and neofunctionalization of a structurally, biochemically, and functionally distinct GLX systems indicate that viridiplantae are under strong selection to detoxify a diversity of RCS.

Reactive carbonyl species (RCS) such as methylglyoxal (MGO) and glyoxal (GO) are highly reactive, unwanted side-products of cellular metabolism maintained at harmless intracellular levels by specific scavenging mechanisms. MGO and GO are metabolized through the glyoxalase (GLX) system, which consists of two enzymes acting in sequence, GLXI and GLXII. While plant genomes encode a number of different GLX isoforms, their specific functions and how they arose during evolution are unclear. Here, we used Arabidopsis (Arabidopsis thaliana) as a model species to investigate the evolutionary history of GLXI and GLXII in plants and whether the GLX system can protect plant cells from the toxicity of RCS other than MGO and GO. We show that plants possess two GLX systems of different evolutionary origins and with distinct structural and functional properties. The first system is shared by all eukaryotes, scavenges MGO and GO, especially during seedling establishment, and features Zn2+-type GLXI proteins with a metal co-factor preference that were present in the last eukaryotic common ancestor. GLXI and GLXII of the second system, featuring Ni2+-type GLXI, were acquired by the last common ancestor of Viridiplantae through horizontal gene transfer from proteobacteria and can together metabolize keto-D-glucose (KDG, glucosone), a glucose-derived RCS, to D-gluconate. When plants displaying loss-of-function of a Viridiplantae-specific GLXI were grown in KDG, D-gluconate levels were reduced to 10-15% of those in the wild type, while KDG levels showed an increase of 48-67%. In contrast to bacterial GLXI homologs, which are active as dimers, plant Ni2+-type GLXI proteins contain a domain duplication, are active as monomers, and have a modified second active site. The acquisition and neofunctionalization of a structurally, biochemically, and functionally distinct GLX system indicates that Viridiplantae are under strong selection to detoxify diverse RCS.

Dietary phosphate intake in the Western population greatly exceeds the recommended dietary allowance and is linked to enhanced cardiovascular and all-cause mortality. It is unclear whether a chronic high phosphate diet (HPD) causes kidney injury in healthy individuals. Here, we show that feeding a 2% HPD in C57BL/6N mice for one up to six months resulted in hyperphosphatemia, hyperphosphaturia, increased plasma levels of fibroblast growth factor (FGF) 23, and parathyroid hormone (PTH) compared to mice on a 0.8% phosphate diet. Kidney injury was already noted after two months of HPD characterized by loss of proximal tubular (PT) cell polarity, flattened epithelia, disruption of brush border membranes, vacuolization, increased PT cell proliferation, marked interstitial mononuclear infiltration, and progressive accumulation of collagen fibers. HPD increased Stat3 activation and Kim-1 expression in PT epithelial cells and enhanced renal synthesis of chemokines recruiting monocytes and macrophages as well as macrophage related factors. Enhanced recruitment of F4/80

BACKGROUND AND AIMS Routine pathological diagnostics in kidneys are mainly based on semi-quantitative eyeballing. In own former studies, we showed predictive value of precise immune cell quantification in allografts using digital semi-automated techniques. We now aim to achieve fully automated segmentation workflow with CNNs. METHOD Standard routine stains (immuno/histochemistry, immunofluorescence) were digitized (20×) with Metafer, a commercial scanning/imaging platform. Diagnostically relevant anatomical compartments (cortex, medulla, glomeruli, tubuli [proximal/distal/collecting duct], glomerular/peritubular capillaries and nuclei) were manually annotated by use of immunomarkers to generate large data sets on human renal biopsies and nephrectomies. Data were used to train multi-class semantic segmentation CNNs with broad data augmentation to achieve a robustness against staining variances. RESULTS Using Jones-HE stains for multi-class segmentation, a cortex-medulla-extrarenal CNN revealed pixel based hit rates above 97.9%, detection of glomeruli had a pixel based hit rate above 99%, a multi-class CNN for tubules, tubular membranes and peritubular capillaries resulted in a hit rate of 91.5%, and nuclear-based cell detection shows pixel based hit rates above 98%. Identification of cell location in interstitium, tubuli, glomeruli, peritubular and glomerular capillaries reached very high hit rates: Glomerular endothelial cells actually result in 83% true positives, 13% false negatives and 4% false positives. Additionally, a tubulus classifier (proximal tubulus, distal tubulus, collecting duct and atrophic tubulus) with an accuracy >90% was developed. CONCLUSION Automated structure segmentation by CNNs can complement and specify classical nephropathological diagnostics, especially for spatial risk marker evaluation in early transplant biopsies.

BACKGROUND AND AIMS Dietary phosphate intake greatly exceeds the recommended daily allowance in the Western population. Elevated phosphate levels are linked to increased cardiovascular and all-cause mortality, impaired bone health and premature ageing. The renal effects of chronic high dietary phosphate intake for healthy individuals are still not clear. METHOD Male C57BL/6 mice received a 2% high phosphate diet (HPD) or a 0.8% normal phosphate diet (NPD) for 1 to 6 months. We collected blood, urine and kidneys to investigate phosphate metabolism and progression of kidney injury. In vitro analysis for the toxic effects of phosphate or the phosphaturic hormones parathyroid hormone (PTH) and fibroblast growth factor (FGF) 23 was done in HK-2 humane proximal tubule (PT) cells. RESULTS HPD in mice caused hyperphosphataemia despite increased phosphaturia. Both intact FGF23 and PTH plasma levels were significantly increased in mice on HPD at 1 month onward. Mice on HPD showed significantly increased creatinine levels and albuminuria. Histopathological analysis of the kidneys revealed increased PT injury in mice receiving HPD compared to NPD controls from 2 months onward followed by a rapid progression of organ damage from month 5 to 6. Picrosirius red staining of kidney sections showed a progressively increased accumulation of collagen fibers in renal cortex of mice on HPD, which was significant from 4 months onward and accompanied by enhanced expression of transforming growth factor-β1, collagen 1 and fibronectin. The significant induction of the tubular injury marker Havcr1, encodes for Kim-1, started already after 1 month of feeding HPD, progressed over time and was strongest after 6 months. Immunofluorescence staining revealed Kim-1 localization to damaged PT, which was most prominent after 3 and 6 months of HPD. Renal Kim-1 protein levels were positively associated with tubular injury score and tubulointerstitial fibrosis. Histological staining and quantification revealed significantly increased number of pStat3+ cells in PTs of HPD-fed mice at months 1, 2, 5 and 6, indicating activated Stat3 signaling. The number of pStat3+ cells was positively associated with Kim-1 synthesis. Inflammation cytokine array with kidney tissue lysates showed, among others, enhanced expression of monocyte chemoattractant protein (MCP)-1 and macrophage colony-stimulating factor (MCSF) in HPD-fed mice. The mRNA expression of Csf1 (encodes for MCSF) was upregulated in HPD-fed mice and most pronounced after 6 months. Ccl2 mRNA expression (encodes for MCP-1) was significantly enhanced after 3, 5 and 6 months of HPD compared to NPD. Immunofluorescence staining showed a strong and specific induction of MCP-1 in PT epithelial cells due to HPD. The HPD-induced Ccl2 expression was positively associated with Kim-1 expression indicating an interaction between pStat3/Kim-1 signalling pathway and MCP-1. Immunohistochemical staining and flow cytometry analysis revealed a significantly increased recruitment of F4/80+ macrophages in renal tissue of HPD-fed mice after 3 and 6 months. The enhanced renal macrophage recruitment in the entire HPD cohort was associated with increased MCP-1 protein synthesis and increased tubular injury score. In vitro experiments using HK-2 cells showed that stimulation with FGF23 or high phosphate, but not PTH, induced the phosphorylation of STAT3. Interestingly, only treatment with high phosphate significantly upregulated HAVCR1 and CCL2 expression. CONCLUSION Chronic oral phosphate load in mice induced progressive inflammatory kidney injury dominated by macrophages. The direct toxic effects of phosphate are mediated by Stat3/Kim-1 signalling activation in PT cells. Our data support the hypothesis that kidney injury induced by widespread high dietary phosphate intake may cause a global health problem and underline the urgent need for clinical studies on this issue.

Renal immune cells serve as sentinels against ascending bacteria but also promote detrimental inflammation. The kidney medulla is characterized by extreme electrolyte concentrations. We here address how its main osmolytes, NaCl and urea, regulate tubular cell cytokine expression and monocyte chemotaxis. In the healthy human kidney, more monocytes were detected in medulla than cortex. The monocyte gradient was attenuated in patients with medullary NaCl depletion by loop diuretic therapy and in the nephrotic syndrome. Renal tubular epithelial cell gene expression responded similarly to NaCl and tonicity control mannitol, but not urea. NaCl significantly upregulated chemotactic cytokines, most markedly CCL26, CCL2, and CSF1. This induction was inhibited by the ROS scavenger n-acetylcysteine. In contrast, urea, the main medullary osmolyte in catabolism, dampened tubular epithelial CCL26 and CSF1 expression. Renal medullary chemokine and monocyte marker expression decreased in catabolic mice. NaCl-, but not urea-stimulated tubular epithelium or CCL2 and CCL26, promoted human classical monocyte migration. CCL26 improved bactericidal function. In the human kidney medulla, monocyte densities correlated with tubular CCL26 protein abundance. In summary, medullary-range NaCl, but not urea, promotes tubular cytokine expression and monocyte recruitment. This may contribute to the pyelonephritis vulnerability in catabolism but can possibly be harnessed against pathologic inflammation.

Ferroptosis, a type of iron-dependent programmed cell death distinct from apoptosis, necroptosis, and other types of cell death, is characterized by lipid peroxidation, reactive oxygen species production, and mitochondrial dysfunction. Accumulating evidence has highlighted vital roles for ferroptosis in multiple diseases, including acute kidney injury. Therefore, ferroptosis has become a major focus for translational research. However, despite its involvement in pathological conditions, there are no pharmacologic inhibitors of ferroptosis in clinical use. In the context of drug repurposing, a strategy for identifying new uses for approved drugs outside the original medical application, we discovered that vitamin K1 is an efficient inhibitor of ferroptosis. Our findings are strengthened by the fact that the vitamin K antagonist phenprocoumon significantly exacerbated ferroptotic cell death in vitro and also massively worsened the course of acute kidney injury in vivo, which is of utmost clinical importance. We therefore assign vitamin K1 a novel role in preventing ferroptotic cell death in acute tubular necrosis during acute kidney injury. Since the safety, tolerability, pharmacokinetics, and pharmacodynamics of vitamin K1 formulations are well documented, this drug is primed for clinical application, and provides a new strategy for pharmacological control of ferroptosis and diseases associated with this mode of cell death.

Interstitial fibrosis and tubular atrophy, a major cause of kidney allograft dysfunction, has been linked to premature cellular senescence. The mTOR inhibitor Rapamycin protects from senescence in experimental models, but its antiproliferative properties have raised concern early after transplantation particularly at higher doses. Its effect on senescence has not been studied in kidney transplantation, yet. Rapamycin was applied to a rat kidney transplantation model (3 mg/kg bodyweight loading dose, 1.5 mg/kg bodyweight daily dose) for 7 days. Low Rapamycin trough levels (2.1-6.8 ng/mL) prevented the accumulation of p16(INK4a) positive cells in tubules, interstitium, and glomerula. Expression of the cytokines MCP-1, IL-1 beta, and TNF-alpha, defining the proinflammatory senescence-associated secretory phenotype, was abrogated. Infiltration with monocytes/macrophages and CD8(+) T-lymphocytes was reduced and tubular function was preserved by Rapamycin. Inhibition of mTOR was not associated with impaired structural recovery, higher glucose levels, or weight loss. mTOR inhibition with low-dose Rapamycin in the immediate posttransplant period protected from premature cellular senescence without negative effects on structural and functional recovery from preservation/reperfusion damage, glucose homeostasis, and growth in a rat kidney transplantation model. Reduced senescence might maintain the renal regenerative capacity rendering resilience to future injuries resulting in protection from interstitial fibrosis and tubular atrophy.

Background To date, there is insufficient knowledge about crescentic glomerulonephritis (cGN), the most frequent immunologic cause of acute kidney injury in children. Methods Over a period of 16 years, we retrospectively analyzed kidney biopsy results, the clinical course, and laboratory data in 60 pediatric patients diagnosed with cGN. Results The underlying diseases were immune complex GN (n = 45/60, 75%), including IgA nephropathy (n = 19/45, 42%), lupus nephritis (n = 10/45, 22%), Henoch-Schoenlein purpura nephritis (n = 7/45, 16%) and post-infectious GN (n = 7/45, 16%), ANCA-associated pauci-immune GN (n = 10/60, 17%), and anti-glomerular basement-membrane GN (n = 1/60, 2%). Patient CKD stages at time of diagnosis and at a median of 362 days (range 237–425) were CKD I: n = 13/n = 29, CKD II: n = 15/n = 9, CKD III: n = 16/n = 7, CKD IV: n = 3/n = 3, CKD V: n = 13/n = 5. Course of cGN was different according to class of cGN, duration of disease from first clinical signs to diagnosis of cGN by biopsy, percentage of crescentic glomeruli, amount of tubular atrophy/interstitial fibrosis and necrosis on renal biopsy, gender, age, nephrotic syndrome, arterial hypertension, dialysis at presentation, and relapse. Forty-eight/60 children were treated with ≥ 5 (methyl-) prednisolone pulses and 53 patients received oral prednis(ol)one in combination with mycophenolate mofetil (n = 20), cyclosporine A (n = 20), and/or cyclophosphamide (n = 6), rituximab (n = 5), azathioprine (n = 2), tacrolimus (n = 1), and plasmapheresis/immunoadsorption (n = 5). Conclusions The treatment success of cGN is dependent on early diagnosis and aggressive therapy, as well as on the percentage of crescentic glomeruli on renal biopsy and on the underlying type of cGN. CsA and MMF seem to be effective alternatives to cyclophosphamide.

Arabidopsis plants overexpressing glycolate oxidase in chloroplasts (GO5) and loss-of-function mutants of the major peroxisomal catalase isoform, cat2-2, produce increased hydrogen peroxide (H2 O2 ) amounts from the respective organelles when subjected to photorespiratory conditions like increased light intensity. Here, we have investigated if and how the signaling processes triggered by H2 O2 production in response to shifts in environmental conditions and the concomitant induction of indole phytoalexin biosynthesis in GO5 affect susceptibility towards the hemibiotrophic fungus Colletotrichum higginsianum. Combining histological, biochemical, and molecular assays, we found that the accumulation of the phytoalexin camalexin was comparable between GO genotypes and cat2-2 in the absence of pathogen. Compared with wild-type, GO5 showed improved resistance after light-shift-mediated production of H2 O2 , whereas cat2-2 became more susceptible and allowed significantly more pathogen entry. Unlike GO5, cat2-2 suffered from severe oxidative stress after light shifts, as indicated by glutathione pool size and oxidation state. We discuss a connection between elevated oxidative stress and dampened induction of salicylic acid mediated defense in cat2-2. Genetic analyses demonstrated that induced resistance of GO5 is dependent on WRKY33, but not on camalexin production. We propose that indole carbonyl nitriles might play a role in defense against C. higginsianum.

The role of endothelial cells in the pathophysiology of antibody-mediated rejection after renal transplantation has been widely investigated. We expand this scenario to the impact of epithelial cells on the microenvironment during rejection. Primary proximal tubular epithelial cells were stimulated via HLA class I, CD155 and CD166 based on their potential signal-transducing capacity to mediate back signaling after encounter with either T/NK cells or donor-specific antibodies. Upon crosslinking of these ligands with mAbs, PTEC secreted IL-6, CXCL1,8,10, CCL2, and sICAM-1. These proteins were also released by PTEC as consequence of a direct interaction with T/NK cells. Downmodulation of the receptor CD226 on effector cells confirmed the involvement of this receptor/ligand pair in back signaling. In vivo, CD155 and CD166 expression was detectable in proximal and distal tubuli of renal transplant biopsies, respectively. The composition of the protein microenvironment in these biopsies showed a substantial overlap with the PTEC response. Cluster and principal component analyses of the microenvironment separated unsuspicious from rejection biopsies and, furthermore, ABMR, TCMR, and borderline rejection. In conclusion, our results provide evidence that epithelial cells may contribute to the rejection process and pave the way to a better understanding of the pathomechanisms of kidney allograft rejection.

Fibrosis is a progressive biological condition, leading to organ dysfunction in various clinical settings. Although fibroblasts and macrophages are known as key cellular players for fibrosis development, a comprehensive functional model that considers their interaction in the metabolic/immunologic context of fibrotic tissue has not been set up. Here we show, by transcriptome-based mathematical modeling in an in vitro system that represents macrophage-fibroblast interplay and reflects the functional effects of inflammation, hypoxia and the adaptive immune context, that irreversible fibrosis development is associated with specific combinations of metabolic and inflammatory cues. The in vitro signatures are in good alignment with transcriptomic profiles generated on laser captured glomeruli and cortical tubule-interstitial area, isolated from human transplanted kidneys with advanced stages of glomerulosclerosis and interstitial fibrosis/tubular atrophy, two clinically relevant conditions associated with organ failure in renal allografts. The model we describe here is validated on tissue based quantitative immune-phenotyping of biopsies from transplanted kidneys, demonstrating its feasibility. We conclude that the combination of in vitro and in silico modeling represents a powerful systems medicine approach to dissect fibrosis pathogenesis, applicable to specific pathological conditions, and develop coordinated targeted approaches.

Delayed graft function (DGF) is a strong risk factor for development of interstitial fibrosis and tubular atrophy (IFTA) in kidney transplants. Quantitative assessment of inflammatory infiltrates in kidney biopsies of DGF patients can reveal predictive markers for IFTA development. In this study, we combined multiplex tyramide signal amplification (mTSA) and convolutional neural networks (CNNs) to assess the inflammatory microenvironment in kidney biopsies of DGF patients (n = 22) taken at 6 weeks post-transplantation. Patients were stratified for IFTA development (, This study describes a methodology to assess the microenvironment in sparse tissue samples. Deep learning, multiplex immunohistochemistry, and mathematical image processing techniques were incorporated to quantify lymphocytes, macrophages, and capillaries in kidney transplant biopsies of delayed graft function patients. The quantitative results were used to assess correlations with development of interstitial fibrosis and tubular atrophy.

Background and Aims Macrophages and monocytes are main players in innate immunity. In renal diseases, their role is poorly understood. Our multicentric cross-sectional study aimed to study the prevalence of macrophages and monocytes in various human native kidney diseases. For this, we used precise pixel-based digital quantification of their densities in renal biopsies and correlated our findings with clinical data. Method We included 324 patients, who underwent a diagnostic renal biopsy. Additional normal kidney samples from 16 tumour nephrectomies were used as controls. According to the diagnosed diseases, we established 17 patient groups. Biopsies were stained for CD68+-macrophages using automated immunohistochemistry (Ventana Ultra) and selected groups were further subtyped for CD14+-monocytes and CD163+-M2-macrophages (67 cases, pauci-immune glomerulonephritis (PIGN), IgA-nephropathy (IgAN) and control samples). Digitized sections (Leica) were analysed using the open-source software QuPath to quantify cell densities (positively stained areas displayed as percentages of ROI) in renal cortex, medulla and extrarenal tissue, respectively. Detailed clinical and laboratory data at timepoint of biopsy were available for all patients. Additional data for follow-up were achievable in 158 cases. Results Renal disease samples presented higher mean macrophage densities compared to control cases (CD68: cortex 1.2 vs. 0.2%, p64 years) showed a higher medullary M2-infiltration (1.81% vs. 4.34%, p Conclusion Macrophages may promote progression of human renal diseases, whereas monocytes do not correlate with eGFR-decline. Especially, in cANCA- vasculitis CD163+- infiltration is associated with renal outcome. Additional studies are needed to investigate, whether macrophages can serve as predictive markers or therapeutical targets in native renal diseases.

Summary Kidney injury significantly increases overall mortality. Neutrophilic granulocytes (neutrophils) are the most abundant human blood leukocytes. They are characterized by a high turnover rate, chiefly controlled by granulocyte colony stimulating factor (G‐CSF). The role of kidney injury and uremia in regulation of granulopoiesis has not been reported. Kidney transplantation, which inherently causes ischemia–reperfusion injury of the graft, elevated human neutrophil expression of the surface glycoprotein CD177. CD177 is among the most G‐CSF‐responsive neutrophil genes and reversibly increased on neutrophils of healthy donors who received recombinant G‐CSF. In kidney graft recipients, a transient rise in neutrophil CD177 correlated with renal tubular epithelial G‐CSF expression. In contrast, CD177 was unaltered in patients with chronic renal impairment and independent of renal replacement therapy. Under controlled conditions of experimental ischemia–reperfusion and unilateral ureteral obstruction injuries in mice, renal G‐CSF mRNA and protein expression significantly increased and systemic neutrophilia developed. Human renal tubular epithelial cell G‐CSF expression was promoted by hypoxia and proinflammatory cytokine interleukin 17A in vitro. Clinically, recipients of ABO blood group‐incompatible kidney grafts developed a larger rise in neutrophil CD177. Their grafts are characterized by complement C4d deposition on the renal endothelium, even in the absence of rejection. Indeed, complement activation, but not hypoxia, induced primary human endothelial cell G‐CSF expression. Our data demonstrate that kidney injury induces renal G‐CSF expression and modulates granulopoiesis. They delineate differential G‐CSF regulation in renal epithelium and endothelium. Altered granulopoiesis may contribute to the systemic impact of kidney injury., Our prospective study in kidney graft recipients shows that human neutrophil CD177, a major G‐CSF target gene, significantly and reversibly increases after transplantation. Two mouse models demonstrate enhanced renal G‐CSF expression and granulopoiesis after acute kidney injury under controlled conditions in vivo. Experiments in primary human cells revealed differential regulation of G‐CSF in human renal tubular epithelium and vascular endothelium by hypoxia, IL‐17A and complement activation as possible underlying mechanisms.

Zusammenfassung Einleitung Bei Patientinnen mit Status nach Sectio ist die sonografische Diagnostik des unteren Uterinsegments (uUS) weit verbreitet. Ihr Nutzen für die Geburtsplanung ist jedoch nicht ausreichend belegt. Es gibt weder ein einheitliches Untersuchungsprotokoll noch einen sicheren Cut-off-Wert. Ziel dieser Studie war es, den Wert der uUS-Diagnostik in der klinischen Routine zu prüfen. Material und Methoden In die retrospektive Analyse wurden 631 Patientinnen mit Status nach Sectio eingeschlossen. Bei 399 (63%) Patientinnen war eine sonografische Dickenmessung des Myometriums (muUS) und/oder des gesamten uUS (guUS)) erfolgt. Die Inzidenz von Uterusdefekten sowie der Zeitpunkt und die Modalität ihrer Diagnostik wurden eruiert. Sensitivitäten, Spezifitäten, positive (PPV) und negative prädiktive Werte (NPV) wurden für verschiedene Cut-offs errechnet. Ergebnisse Uterusdefekte traten bei 28 (4,4%) Patientinnen auf. Die Detektionsrate für die Sonografie war gering (13,6%), die Sensitivität/ Spezifität/ PPV/ NPV für niedrigere Cut-off-Werte (2 mm (guUS)/ 1 mm (muUS): 75/96/48,3/98,7%) jedoch besser als für höhere (3 mm (guUS)/ 2 mm (muUS): 15,8/93,2/10,4/68,7%). Diskussion Der Nutzen der sonografischen uUS-Diagnostik erscheint überschätzt. Da ihre Genauigkeit von zahlreichen methodischen Faktoren beeinflusst wird, könnte sie durch ein einheitliches Protokoll mit einem transabdominellen und transvaginalen Scan durch einen erfahrenen Untersucher verbessert werden. Ein geeigneteres Untersuchungsziel für die Selektion nach Sectio könnte der Ausschluss von Uterusdefekten sein.

Background The etiology of steroid-resistant nephrotic syndrome, which manifests as FSGS, is not completely understood. Aberrant glycosylation is an often underestimated factor for pathologic processes, and structural changes in the glomerular endothelial glycocalyx have been correlated with models of nephrotic syndrome. Glycans are frequently capped by sialic acid (Sia), and sialylation’s crucial role for kidney function is well known. Human podocytes are highly sialylated; however, sialylation’s role in podocyte homeostasis remains unclear. Methods We generated a podocyte-specific sialylation-deficient mouse model ( PCmas −/− ) by targeting CMP-Sia synthetase, and used histologic and ultrastructural analysis to decipher the phenotype. We applied CRISPR/Cas9 technology to generate immortalized sialylation-deficient podocytes (asialo-podocytes) for functional studies. Results Progressive loss of sialylation in PCmas −/− mice resulted in onset of proteinuria around postnatal day 28, accompanied by foot process effacement and loss of slit diaphragms. Podocyte injury led to severe glomerular defects, including expanded capillary lumen, mesangial hypercellularity, synechiae formation, and podocyte loss. In vivo , loss of sialylation resulted in mislocalization of slit diaphragm components, whereas podocalyxin localization was preserved. In vitro , asialo-podocytes were viable, able to proliferate and differentiate, but showed impaired adhesion to collagen IV. Conclusions Loss of cell-surface sialylation in mice resulted in disturbance of podocyte homeostasis and FSGS development. Impaired podocyte adhesion to the glomerular basement membrane most likely contributed to disease development. Our data support the notion that loss of sialylation might be part of the complex process causing FSGS. Sialylation, such as through a Sia supplementation therapy, might provide a new therapeutic strategy to cure or delay FSGS and potentially other glomerulopathies.

Introduction: Renal cell carcinoma (RCC), an immunogenic tumor, is the most common form of kidney cancer worldwide. Immune checkpoint inhibitors (ICIs) play an important role in the treatment of metastatic RCC. Programmed death-ligand (PD-L1) has already been proposed as a possible prognosticator for ICIs effectiveness. To elucidate the feasible role of ICIs in neoadjuvant settings, we have assessed the most common PD-L1 expression modalities [tumor proportion score (TPS), combined positivity score (CPS) and inflammatory cell (IC) score] in primary tumors (PTs) and venous tumor thrombi (VTT) in first diagnosed, previously untreated RCC patients with accompanying VTT. Methods: Between January 1999 and December 2016, 71 patients with a first diagnosed, untreated, locally advanced RCC (aRCC) (≥ pT3a) underwent surgery in Hanover Medical School (MHH). PD-L1 expression was examined separately in PTs and VTT using the CPS, IC score and TPS. We also considered the age at the time of the initial surgery and gender as probable influencing factors. By using a cutoff value of 1 (1%), PD-L1 expression levels in PTs and VTT were assessed to enable the determination of any frequency differences. Results: Positive scores for PTs were shown by 54 (CPS), 53 (IC score) and 34 (TPS) patients, whereas in VTT, positive scores were evaluated for a total of 50 (CPS), 47 (IC-score) and 36 (TPS) patients. No statistically significant differences were obtained between the PD-L1 expression immunoscores for PTs and VTT. The covariates age at the time of the initial surgery and gender could not be statistically proven to influence the differences in PD-L1 expression between the VTT and PTs. Conclusion: To the best of our knowledge, this research is the largest study to investigate PD-L1 expression in PTs and VTT in 71 cases. It could have relevance for the future development of neoadjuvant immunotherapy options, particularly in aRCC with VTT.

International audience; Objective: This article presents an automatic image processing framework to extract quantitative high-level information describing the micro-environment of glomeruli in consecutive whole slide images (WSIs) processed with different staining modalities of patients with chronic kidney rejection after kidney transplantation.Methods: This four-step framework consists of: 1) approximate rigid registration, 2) cell and anatomical structure segmentation 3) fusion of information from different stainings using a newly developed registration algorithm 4) feature extraction.Results: Each step of the framework is validated independently both quantitatively and qualitatively by pathologists. An illustration of the different types of features that can be extracted is presented.Conclusion: The proposed generic framework allows for the analysis of the micro-environment surrounding large structures that can be segmented (either manually or automatically). It is independent of the segmentation approach and is therefore applicable to a variety of biomedical research questions.Significance: Chronic tissue remodelling processes after kidney transplantation can result in interstitial fibrosis and tubular atrophy (IFTA) and glomerulosclerosis. This pipeline provides tools to quantitatively analyse, in the same spatial context, information from different consecutive WSIs and help researchers understand the complex underlying mechanisms leading to IFTA and glomerulosclerosis

Main conclusion The biochemical characterization of glycolate oxidase in Ricinus communis hints to different physiological functions of the enzyme depending on the organ in which it is active. Abstract Enzymatic activities of the photorespiratory pathway are not restricted to green tissues but are present also in heterotrophic organs. High glycolate oxidase (GOX) activity was detected in the endosperm of Ricinus communis. Phylogenetic analysis of the Ricinus l-2-hydroxy acid oxidase (Rc(l)-2-HAOX) family indicated that Rc(l)-2-HAOX1 to Rc(l)-2-HAOX3 cluster with the group containing streptophyte long-chain 2-hydroxy acid oxidases, whereas Rc(l)-2-HAOX4 clusters with the group containing streptophyte GOX. Rc(l)-2-HAOX4 is the closest relative to the photorespiratory GOX genes of Arabidopsis. We obtained Rc(l)-2-HAOX4 as a recombinant protein and analyze its kinetic properties in comparison to the Arabidopsis photorespiratory GOX. We also analyzed the expression of all Rc(l)-2-HAOXs and conducted metabolite profiling of different Ricinus organs. Phylogenetic analysis indicates that Rc(l)-2-HAOX4 is the only GOX encoded in the Ricinus genome (RcGOX). RcGOX has properties resembling those of the photorespiratory GOX of Arabidopsis. We found that glycolate, the substrate of GOX, is highly abundant in non-green tissues, such as roots, embryo of germinating seeds and dry seeds. We propose that RcGOX fulfills different physiological functions depending on the organ in which it is active. In autotrophic organs it oxidizes glycolate into glyoxylate as part of the photorespiratory pathway. In fast growing heterotrophic organs, it is most probably involved in the production of serine to feed the folate pathway for special demands of those tissues.

Background and Aims In early stages of chronic kidney disease (CKD) rising levels of the phosphaturic hormone fibroblast growth factor (FGF) 23 maintain the phosphate homeostasis, whereas in end-stage kidney disease hyperphosphatemia manifests. Both elevated FGF23 and phosphate levels contribute to CKD progression and are associated with an increased mortality in CKD patients. So far, it is unknown whether high inorganic phosphate uptake causes renal injury as well in the absence of CKD. Therefore, we analyzed the effects of a long-term high phosphate diet (HPD) on the renal function in healthy mice. Method We studied the effects of a 2% HPD compared to a 0.8% normal phosphate diet (NPD) fed to healthy male C57BL/6 mice. After six month, blood pressure and metabolic serum and urine parameters were measured. The renal function was characterized by histology, flow cytometry and gene expression analysis. Additionally, we investigate the effects of elevated FGF23 and phosphate on mouse proximal tubular cells (mPT) in vitro. Results Feeding of a HPD for 6 months significantly increased systolic blood pressure (95 vs 79 mmHg; p=0.003), plasma creatinine (0.33±0.01 vs 0.29±0.02 mg/dL; p=0.004), C-term FGF23 (1991±394 vs 309±56 pg/mL; p Conclusion Chronic high phosphate load induces kidney injury in the proximal tubules with severe inflammation and fibrosis in healthy mice. Our results suggest that a controlled dietary intake of inorganic phosphate is not only important for CKD patients but also for the general population.

Aims Monocytes are central for atherosclerotic vascular inflammation. The human non-classical, patrolling subtype, which expresses high levels of CD16 and fractalkine receptor CX3CR1, strongly associates with cardiovascular events. This is most marked in renal failure, a condition with excess atherosclerosis morbidity. The underlying mechanism is not understood. This study investigated how human CD16+ monocytes modulate endothelial cell function. Methods and results In patients with kidney failure, CD16+ monocyte counts were elevated and dynamically decreased within a year after transplantation, chiefly due to a drop in CD14+CD16+ cells. The CX3CR1 ligand CX3CL1 was similarly elevated in the circulation of humans and mice with renal impairment. CX3CL1 upregulation was also observed close to macrophage rich human coronary artery plaques. To investigate a mechanistic basis of this association, CD16+CX3CR1HIGH monocytes were co-incubated with primary human endothelium in vitro. Compared to classical CD14+ monocytes or transwell cocultures, CD16+ monocytes enhanced endothelial STAT1 and NFκB p65 phosphorylation, upregulated expression of CX3CL1 and IL-1β, numerous CCL and CXCL chemokines and molecules promoting leukocyte patrolling and adhesion such as ICAM1 and VCAM1. Genes required for vasodilatation including eNOS decreased while endothelial collagen production increased. Uremic patients' monocytes enhanced endothelial CX3CL1 even more markedly. Their receptor CX3CR1 was required for enhanced aortic endothelial stiffness in murine atherosclerosis with renal impairment. CX3CR1 dose-dependently modulated monocyte-contact-dependent gene expression in human endothelium. Conclusions By demonstrating endothelial proatherosclerotic gene regulation in direct contact with CD16+ monocytes, in part via cellular CX3CR1-CX3CL1 interaction, our data delineate a mechanism how this celltype can increase cardiovascular risk. Translational perspective Human CD16+ monocytes strongly associate with cardiovascular disease. Our data show that they induce primary human arterial and venous endothelial chemokine and adhesion molecule expression and increase mediators of vascular stiffness in direct interaction of the endothelium with CX3CR1. CD16+ monocytes reversibly increase in human kidney failure. Our results propose CD16+ monocytes as mediators of cardiovascular events including the reversible vascular phenotype of patients with kidney failure. Interference with their direct contact to the endothelium may represent a causal therapeutic approach in this high-risk patient group.

Supplemental Digital Content is available in the text., Background. Systemic exposure to high-dose corticosteroids effectively combats acute rejection after kidney transplantation, but at the cost of substantial side effects. In this study, a murine acute renal allograft rejection model was used to investigate whether liposomal-encapsulated prednisolone (LP) facilitates local exposure to enhance its therapeutic effect. Methods. Male BalbC recipients received renal allografts from male C57BL/6J donors. Recipients were injected daily with 5 mg/kg cyclosporine A and received either 10 mg/kg prednisolone (P), or LP intravenously on day 0, 3, and 6, or no additional treatment. Functional magnetic resonance imaging (fMRI) was performed on day 6 to study allograft perfusion and organs were retrieved on day 7 for further analysis. Results. Staining of polyethylene-glycol-labeled liposomes and high performance liquid chromatography analysis revealed accumulation in the LP treated allograft. LP treatment induced the expression of glucocorticoid responsive gene Fkbp5 in the allograft. Flow-cytometry of allografts revealed liposome presence in CD45+ cells, and reduced numbers of F4/80+ macrophages, and CD3+ T-lymphocytes upon LP treatment. Banff scoring showed reduced interstitial inflammation and tubulitis and fMRI analysis revealed improved allograft perfusion in LP versus NA mice. Conclusions. Liposomal delivery of prednisolone improved renal bio-availability, increased perfusion and reduced cellular infiltrate in the allograft, when compared with conventional prednisolone. Clinical studies should reveal if treatment with LP results in improved efficacy and reduced side effects in patients with renal allograft rejection.

Supplemental Digital Content is available in the text., Background: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. Methods: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II–mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. Results: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. Conclusions: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.

Acute tubular injury (ATI) is common in renal allografts and is related to inferior long-term allograft function. However, it is unknown which of the morphological features of ATI can predict outcome and how they should be graded. Here, we examine features of ATI systematically in protocol biopsies and biopsies for cause to define the most predictive features. Analyses included 521 protocol biopsies taken at 6 wk, 3 mo, and 6 mo after transplantation and 141 biopsies for cause from 204 patients. Features of ATI included brush border loss, tubular epithelial lucency, flattening, pyknosis, nuclei loss, and luminal debris, each graded semiquantitatively. Additional immunohistochemical stainings were performed for markers of cell injury (neutrophil gelatinase-associated lipocalin), cell death [cleaved caspase-3, fatty acid-coenzyme A ligase 4 (FACL4)], and proliferation (Ki-67). Interobserver reliability was good for pyknosis, flattening, and brush border loss and poor for lucency, nuclei loss, and luminal debris. In protocol biopsies between 6 wk and 6 mo, the degree of ATI remained virtually unchanged. Biopsies for cause had generally higher injury scores. Deceased donor source, delayed graft function, ganciclovir/valganciclovir treatment, and urinary tract infection correlated with ATI. The degree of pyknosis, flattening, and brush border loss correlated best with impaired allograft function. FACL4 expression was observed in areas of ATI. Only patients with Ki-67 expression showed stable or improved allograft function in the longitudinal assessment. Reliable assessment of ATI is possible by semiquantitative grading of tubular epithelial cell brush border loss, flattening, and pyknosis. Examination of Ki-67 expression can help determine the potential for recovery from this damage.