Hepatitis E virus infection of transplanted kidneys.

Immunocompromised patients are at risk of chronic hepatitis E (HEV) infection. Recurrent T cell and borderline rejections in a pediatric patient with high HEV copy numbers led us to study HEV infection within renal transplants. To investigate the frequency of renal HEV infection in transplanted patients, 15 samples from patients with contemporaneous diagnoses of HEV infection were identified at our center. Ten samples had sufficient residual paraffin tissue for immunofluorescence (IF) and RNA-fluorescence-in-situ-hybridization (RNA-FISH). The biopsy of the pediatric index patient was additionally sufficient for tissue polymerase chain reaction and electron microscopy. HEV RNA was detected in paraffin tissue of the index patient by tissue polymerase chain reaction. Subsequently, HEV infection was localized in tubular epithelial cells by IF, RNA-FISH, and electron microscopy. One additional biopsy from an adult was positive for HEV by RNA-FISH and IF. Focal IF positivity for HEV peptide was observed in 7 additional allografts. Ribavirin therapy was not successful in the pediatric index patient; after relapse, ribavirin is still administered. In the second patient, successful elimination of HEV was achieved after short-course ribavirin therapy. HEV infection is an important differential diagnosis for T cell rejection within transplanted kidneys. Immunostaining of HEV peptide does not necessarily prove acute infection. RNA-FISH seems to be a reliable method to localize HEV.

Inhibition of the renal apical sodium dependent bile acid transporter prevents cholemic nephropathy in mice with obstructive cholestasis.

BACKGROUND & AIMS: Cholemic nephropathy (CN) is a severe complication of cholestatic liver diseases for which there is no specific treatment. We revisited its pathophysiology with the aim of identifying novel therapeutic strategies. METHODS: Cholestasis was induced by bile duct ligation (BDL) in mice. Bile flux in kidneys and livers was visualized by intravital imaging, supported by MALDI mass spectrometry imaging and liquid chromatography-tandem mass spectrometry. The effect of AS0369, a systemically bioavailable apical sodium-dependent bile acid transporter (ASBT) inhibitor, was evaluated by intravital imaging, RNA-sequencing, histological, blood, and urine analyses. Translational relevance was assessed in kidney biopsies from patients with CN, mice with a humanized bile acid (BA) spectrum, and via analysis of serum BAs and KIM-1 (kidney injury molecule 1) in patients with liver disease and hyperbilirubinemia. RESULTS: Proximal tubular epithelial cells (TECs) reabsorbed and enriched BAs, leading to oxidative stress and death of proximal TECs, casts in distal tubules and collecting ducts, peritubular capillary leakiness, and glomerular cysts. Renal ASBT inhibition by AS0369 blocked BA uptake into TECs and prevented kidney injury up to 6 weeks after BDL. Similar results were obtained in mice with humanized BA composition. In patients with advanced liver disease, serum BAs were the main determinant of KIM-1 levels. ASBT expression in TECs was preserved in biopsies from patients with CN, further highlighting the translational potential of targeting ASBT to treat CN. CONCLUSIONS: BA enrichment in proximal TECs followed by oxidative stress and cell death is a key early event in CN. Inhibiting renal ASBT and consequently BA enrichment in TECs prevents CN and systemically decreases BA concentrations. IMPACT AND IMPLICATIONS: Cholemic nephropathy (CN) is a severe complication of cholestasis and an unmet clinical need. We demonstrate that CN is triggered by the renal accumulation of bile acids (BAs) that are considerably increased in the systemic blood. Specifically, the proximal tubular epithelial cells of the kidney take up BAs via the apical sodium-dependent bile acid transporter (ASBT). We developed a therapeutic compound that blocks ASBT in the kidneys, prevents BA overload in tubular epithelial cells, and almost completely abolished all disease hallmarks in a CN mouse model. Renal ASBT inhibition represents a potential therapeutic strategy for patients with CN.

Viridiplantae-specific GLXI and GLXII isoforms co-evolved and detoxify glucosone in planta.

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.

Preoperative Delirium Risk Screening in Patients Undergoing a Cardiac Surgery: Results from the Prospective Observational FINDERI Study.

OBJECTIVE: Postoperative delirium (POD) is a common complication of cardiac surgery that is associated with higher morbidity, longer hospital stay, cognitive decline, and mortality. Preoperative assessments may help to identify patients´ POD risk. However, a standardized screening assessment for POD risk has not been established. DESIGN: Prospective observational FINd DElirium RIsk factors (FINDERI) study. PARTICIPANTS: Patients aged ≥50 years undergoing cardiac surgery. MEASUREMENTS: The primary aim was to analyze the predictive value of the Delirium Risk Screening Questionnaire (DRSQ) prior to cardiac surgery. Secondary aims are to investigate cognitive, frailty, and geriatric assessments, and to use data-driven machine learning (ML) in predicting POD. Predictive properties were assessed using receiver operating characteristics analysis and multivariate approaches (regularized LASSO regression and decision trees). RESULTS: We analyzed a data set of 504 patients (68.3 ± 8.2 years, 21.4% women) who underwent cardiac surgery. The incidence of POD was 21%. The preoperatively administered DRSQ showed an area under the curve (AUC) of 0.68 (95% CI 0.62, 0.73), and the predictive OR was 1.25 (95% CI 1.15, 1.35, p <0.001). Using a ML approach, a three-rule decision tree prediction model including DRSQ (score>7), Trail Making Test B (time>118), and Montreal Cognitive Assessment (score ≤ 22) was identified. The AUC of the three-rule decision tree on the training set was 0.69 (95% CI 0.63, 0.75) and 0.62 (95% CI 0.51, 0.73) on the validation set. CONCLUSION: Both the DRSQ and the three-rule decision tree might be helpful in predicting POD risk before cardiac surgery.

Kidney medullary sodium chloride concentrations induce neutrophil and monocyte extracellular DNA traps that defend against pyelonephritis in vivo.

Urinary tract infections are common. Here, we delineate a role of extracellular DNA trap (ET) formation in kidney antibacterial defense and determine mechanisms of their formation in the hyperosmotic environment of the kidney medulla. ET of granulocytic and monocytic origin were present in the kidneys of patients with pyelonephritis along with systemically elevated citrullinated histone levels. Inhibition of the transcription coregulatory, peptidylarginine deaminase 4 (PAD4), required for ET formation, prevented kidney ET formation and promoted pyelonephritis in mice. ETs predominantly accumulated in the kidney medulla. The role of medullary sodium chloride and urea concentrations in ET formation was then investigated. Medullary-range sodium chloride, but not urea, dose-, time- and PAD4-dependently induced ET formation even in the absence of other stimuli. Moderately elevated sodium chloride promoted myeloid cell apoptosis. Sodium gluconate also promoted cell death, proposing a role for sodium ions in this process. Sodium chloride induced myeloid cell calcium influx. Calcium ion-free media or -chelation reduced sodium chloride-induced apoptosis and ET formation while bacterial lipopolysaccharide amplified it. Autologous serum improved bacterial killing in the presence of sodium chloride-induced ET. Depletion of the kidney sodium chloride gradient by loop diuretic therapy diminished kidney medullary ET formation and increased pyelonephritis severity. Thus, our data demonstrate that ETs may protect the kidney against ascending uropathogenic E. coli and delineate kidney medullary range sodium chloride concentrations as novel inducers of programmed myeloid cell death.

Renal medullary osmolytes NaCl and urea differentially modulate human tubular cell cytokine expression and monocyte recruitment.

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.

High phosphate-induced progressive proximal tubular injury is associated with the activation of Stat3/Kim-1 signaling pathway and macrophage recruitment.

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+ macrophages around injured PT lesions was timely associated with increased Kim-1 synthesis, tubular MCP-1 expression, and degree of PT injury score. Likewise, tubulointerstitial fibrosis was associated with activation of Stat3/Kim-1 signaling pathway. The stimulation of human proximal tubular cells with high phosphate activated Stat3 signaling and induced HAVCR1 and CCL2 expression. We conclude that high phosphate results in progressive proximal tubular injury, indicating that high dietary phosphate intake may affect kidney health and therefore represents an underestimated health problem for the general population.

Vitamin K1 inhibits ferroptosis and counteracts a detrimental effect of phenprocoumon in experimental acute kidney injury.

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.

COVID-19 associated acute transplant failure after AB0-incompatible living donor kidney transplantation - a case report.

INTRODUCTION: Immunosuppressive therapy is associated with an increased risk of severe courses of SARS-CoV-2 infection, with frequently delayed viral clearance. We report a case of an acute kidney transplant failure in persistent SARS-CoV-2 infection in a patient with absolute B-cell depletion after administration of rituximab for AB0-incompatible living donor kidney transplantation. CASE PRESENTATION: A 34-year-old unvaccinated patient is diagnosed with SARS-CoV-2 infection four months after kidney transplantation. With only mild symptoms and an estimated glomerular filtration rate (eGFR) of 44 ml/min/1.73 m2, therapy with molnupiravir was initially given. Within the next eight weeks, transplant biopsies were performed for acute graft failure. These showed acute T-cell rejection with severe acute tubular epithelial damage with only mild interstitial fibrosis and tubular atrophy (BANFF cat. 4 IB), and borderline rejection (BANFF cat. 3). A therapy with prednisolone and intravenous immunoglobulins was performed twice. With unchanged graft failure, the third biopsy also formally showed BANFF cat. 4 IB. However, fluorescence in situ hybridization detected SARS-CoV-2 viruses in large portions of the distal tubules. After nine weeks of persistent COVID-19 disease neither anti-SARS-CoV-2 IgG nor a SARS-CoV-2-specific cellular immune response could be detected, leading to the administration of sotrovimab and remdesivir. Among them, SARS-CoV-2 clearance, detection of IgG, and improvement of graft function were achieved. CONCLUSION: Lack of viral clearance can lead to complications of SARS-CoV-2 infection with atypical manifestations. In kidney transplant patients, before initiating therapy, the differential diagnoses of "rejection" and "virus infection" should be weighed against each other in an interdisciplinary team of nephrologists, infectious diseases specialists and pathologists.

AA-amyloidosis in captive northern tree shrews (Tupaia belangeri).

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.

Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction.

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.

Quantitative assessment of inflammatory infiltrates in kidney transplant biopsies using multiplex tyramide signal amplification and deep learning.

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 (<10% versus ≥10%) from 6 weeks to 6 months post-transplantation, based on histopathological assessment by three kidney pathologists. One mTSA panel was developed for visualization of capillaries, T- and B-lymphocytes and macrophages and a second mTSA panel for T-helper cell and macrophage subsets. The slides were multi spectrally imaged and custom-made python scripts enabled conversion to artificial brightfield whole-slide images (WSI). We used an existing CNN for the detection of lymphocytes with cytoplasmatic staining patterns in immunohistochemistry and developed two new CNNs for the detection of macrophages and nuclear-stained lymphocytes. F1-scores were 0.77 (nuclear-stained lymphocytes), 0.81 (cytoplasmatic-stained lymphocytes), and 0.82 (macrophages) on a test set of artificial brightfield WSI. The CNNs were used to detect inflammatory cells, after which we assessed the peritubular capillary extent, cell density, cell ratios, and cell distance in the two patient groups. In this cohort, distance of macrophages to other immune cells and peritubular capillary extent did not vary significantly at 6 weeks post-transplantation between patient groups. CD163+ cell density was higher in patients with ≥10% IFTA development 6 months post-transplantation (p < 0.05). CD3+CD8-/CD3+CD8+ ratios were higher in patients with <10% IFTA development (p < 0.05). We observed a high correlation between CD163+ and CD4+GATA3+ cell density (R = 0.74, p < 0.001). Our study demonstrates that CNNs can be used to leverage reliable, quantitative results from mTSA-stained, multi spectrally imaged slides of kidney transplant biopsies.

Targeting muscle-enriched long non-coding RNA H19 reverses pathological cardiac hypertrophy.

AIMS: Pathological cardiac remodelling and subsequent heart failure represents an unmet clinical need. Long non-coding RNAs (lncRNAs) are emerging as crucial molecular orchestrators of disease processes, including that of heart diseases. Here, we report on the powerful therapeutic potential of the conserved lncRNA H19 in the treatment of pathological cardiac hypertrophy. METHOD AND RESULTS: Pressure overload-induced left ventricular cardiac remodelling revealed an up-regulation of H19 in the early phase but strong sustained repression upon reaching the decompensated phase of heart failure. The translational potential of H19 is highlighted by its repression in a large animal (pig) model of left ventricular hypertrophy, in diseased human heart samples, in human stem cell-derived cardiomyocytes and in human engineered heart tissue in response to afterload enhancement. Pressure overload-induced cardiac hypertrophy in H19 knock-out mice was aggravated compared to wild-type mice. In contrast, vector-based, cardiomyocyte-directed gene therapy using murine and human H19 strongly attenuated heart failure even when cardiac hypertrophy was already established. Mechanistically, using microarray, gene set enrichment analyses and Chromatin ImmunoPrecipitation DNA-Sequencing, we identified a link between H19 and pro-hypertrophic nuclear factor of activated T cells (NFAT) signalling. H19 physically interacts with the polycomb repressive complex 2 to suppress H3K27 tri-methylation of the anti-hypertrophic Tescalcin locus which in turn leads to reduced NFAT expression and activity. CONCLUSION: H19 is highly conserved and down-regulated in failing hearts from mice, pigs and humans. H19 gene therapy prevents and reverses experimental pressure-overload-induced heart failure. H19 acts as an anti-hypertrophic lncRNA and represents a promising therapeutic target to combat pathological cardiac remodelling.

The genome of Ricinus communis encodes a single glycolate oxidase with different functions in photosynthetic and heterotrophic organs.

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. 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.

Reactive oxygen species dosage in Arabidopsis chloroplasts can improve resistance towards Colletotrichum higginsianum by the induction of WRKY33.

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.

Cholemic Nephropathy Causes Acute Kidney Injury and Is Accompanied by Loss of Aquaporin 2 in Collecting Ducts.

Impairment of renal function often occurs in patients with liver disease. Hepatorenal syndrome is a significant cause of acute kidney injury (AKI) in patients with cirrhosis (HRS-AKI, type 1). Causes of non-HRS-AKI include cholemic nephropathy (CN), a disease that is characterized by intratubular bile casts and tubular injury. As data on patients with CN are obtained primarily from case reports or autopsy studies, we aimed to investigate the frequency and clinical course of CN. We identified 149 patients who underwent kidney biopsy between 2000 and 2016 at the Department of Gastroenterology, Hepatology and Endocrinology at Hannover Medical School. Of these, 79 had a history of liver disease and deterioration of renal function. When applying recent European Association for the Study of the Liver criteria, 45 of 79 patients (57%) presented with AKI, whereas 34 patients (43%) had chronic kidney disease (CKD). Renal biopsy revealed the diagnosis of CN in 8 of 45 patients with AKI (17.8%), whereas none of the patients with CKD was diagnosed with CN. Univariate analysis identified serum bilirubin, alkaline phosphatase, and urinary bilirubin and urobilinogen as predictive factors for the diagnosis of CN. Histological analysis of AKI patients with normal bilirubin, elevated bilirubin, and the diagnosis of CN revealed loss of aquaporin 2 (AQP2) expression in collecting ducts in patients with elevated bilirubin and CN. Biopsy-related complications requiring medical intervention occurred in 4 of 79 patients (5.1%). Conclusion: CN is a common finding in patients with liver disease, AKI, and highly elevated bilirubin. Loss of AQP2 in AKI patients with elevated bilirubin and CN might be the result of toxic effects of cholestasis and in part be responsible for the impairment of renal function.

Defense against Reactive Carbonyl Species Involves at Least Three Subcellular Compartments Where Individual Components of the System Respond to Cellular Sugar Status.

Methylglyoxal (MGO) and glyoxal (GO) are toxic reactive carbonyl species generated as by-products of glycolysis. The pre-emption pathway for detoxification of these products, the glyoxalase (GLX) system, involves two consecutive reactions catalyzed by GLXI and GLXII. In Arabidopsis thaliana, the GLX system is encoded by three homologs of GLXI and three homologs of GLXII, from which several predicted GLXI and GLXII isoforms can be derived through alternative splicing. We identified the physiologically relevant splice forms using sequencing data and demonstrated that the resulting isoforms have different subcellular localizations. All three GLXI homologs are functional in vivo, as they complemented a yeast GLXI loss-of-function mutant. Efficient MGO and GO detoxification can be controlled by a switch in metal cofactor usage. MGO formation is closely connected to the flux through glycolysis and through the Calvin Benson cycle; accordingly, expression analysis indicated that GLXI is transcriptionally regulated by endogenous sugar levels. Analyses of Arabidopsis loss-of-function lines revealed that the elimination of toxic reactive carbonyl species during germination and seedling establishment depends on the activity of the cytosolic GLXI;3 isoform. The Arabidopsis GLX system involves the cytosol, chloroplasts, and mitochondria, which harbor individual components that might be used at specific developmental stages and respond differentially to cellular sugar status.

Crescentic glomerulonephritis in children.

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.

Podocyte-Specific Sialylation-Deficient Mice Serve as a Model for Human FSGS.

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.

AMYLOIDOSIS IN CARACALS (CARACAL CARACAL).

Nine cases of amyloidosis in caracals (Caracal caracal) from three different institutions in Europe were reviewed and evaluated histopathologically. The six males and three females died between 2008 and 2018 at an age of 6 yr ± 2.5 mo (median ± interquartile range). In two out of nine (2/9) animals, amyloidosis was an incidental postmortem finding; the animals died of bronchopneumonia and gastric ulceration due to Helicobacter spp., respectively. Seven (7/9) animals suffered from acute renal failure due to amyloidosis, one of them additionally of cardiac decompensation. The predominant clinical signs were weight loss, lethargy, dys- or anorexia, dehydration, increased BUN and creatinine, and azotemia. The main gross lesion was a pale renal cortex on cut surface; in two animals, the kidneys appeared enlarged. Histologically, glomerular amyloid was present in every animal (9/9), and was the predominant renal manifestation of amyloidosis. Additional findings included splenic amyloid (8/8), amyloid in the lamina propria of the intestine (5/5), and amyloid in the lingual submucosa (4/4). Gastric mineralization was present in four animals suffering from renal failure. In the animal dying from bronchopneumonia, severe pancreatic amyloid deposits mainly affecting the exocrine pancreas (1/5) were identified. Immunohistochemistry was employed to identify amyloid AA in eight cases; only in the caracal dying from bronchopneumonia AA was amyloid confirmed. In several organs, especially in those where only small amyloid deposits were detected, a Congo red stain was often necessary to confirm the deposition. The etiology of the amyloidosis remains unknown. Three caracals were related within two generations, another three within four generations, so one might hypothesize a familial trait. In conclusion, amyloidosis should be considered as a significant disease in the caracal. Particularly in cases with renal disease, it should be included as a major differential diagnosis.