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Keratins are the main components of the cell cytoskeleton of epithelial cells. Epithelial cells under stressful stimuli react by modifying their keratin expression pattern. Glomerular diseases are pathological conditions that may lead to loss of kidney function if not timely diagnosed and treated properly. This study aims to examine glomerular and tubular keratin expression in podocytopathies, ANCA-associated vasculitis, and IgA nephropathy and how this expression correlates to clinical outcomes. We included 45 patients with podocytopathies (minimal change disease and focal segmental glomerulosclerosis), ANCA-associated vasculitis, and IgA nephropathy, with or without crescentic lesions, and healthy controls. All tissues were assessed by photon microscopy and immunohistochemistry. Biopsy sections were examined for keratins 7, 8, 18, and 19 expression in the glomerular and tubulointerstitial areas separately. Moreover, we examined how keratin expression was correlated with long-term kidney function outcomes. All four studied keratins had significantly increased glomerular expression in patients with ANCA vasculitis compared to controls and MCD patients. Tubular expression of keratins 7, 8, and 19 was related to kidney outcome in all groups. Patients with crescents had higher expression of all keratins in both glomeruli and tubulointerstitium. The presence of tubular atrophy, interstitial fibrosis, mesangial hyperplasia, and interstitial inflammation did not affect keratin expression. Keratins, an abundant component of renal epithelial cells, have the potential to be featured as a biomarker for kidney function prognosis in patients with glomerular diseases. [ABSTRACT FROM AUTHOR]

Chronic kidney diseases affect a substantial percentage of the adult population worldwide. This observation emphasizes the need for novel insights into the molecular mechanisms that control the onset and progression of renal diseases. Recent advances in genomics have uncovered a previously unanticipated link between the non‐coding genome and human kidney diseases. Here we screened and analysed long non‐coding RNAs (lncRNAs) previously identified in mouse kidneys by genome‐wide transcriptomic analysis, for conservation in humans and differential expression in renal tissue from healthy and diseased individuals. Our data suggest that LINC01187 is strongly down‐regulated in human kidney tissues of patients with diabetic nephropathy and rapidly progressive glomerulonephritis, as well as in murine models of kidney diseases, including unilateral ureteral obstruction, nephrotoxic serum‐induced glomerulonephritis and ischemia/reperfusion. Interestingly, LINC01187 overexpression in human kidney cells in vitro inhibits cell death indicating an anti‐apoptotic function. Collectively, these data suggest a negative association of LINC01187 expression with renal diseases implying a potential protective role. [ABSTRACT FROM AUTHOR]

Donor organ-shortage has resulted in the increased use of marginal grafts; however, normothermic machine perfusion (NMP) holds the potential for organ viability assessment and restoration of marginal grafts prior to transplantation. Additionally, cell-, oxygen carrier-free and antioxidants-supplemented solutions could potentially prevent adverse effects (transfusion reactions, inflammation, hemolysis), associated with the use of autologous packed red blood cell (pRBC)-based perfusates. This study compared 6 h NMP of porcine kidneys, using an established pRBC-based perfusate (pRBC, n = 7), with the novel cell- and oxygen carrier-free organ preservation solution Ecosol, containing taurine (Ecosol, n = 7). Despite the enhanced tissue edema and tubular injury in the Ecosol group, related to a suboptimal molecular mass of polyethylene glycol as colloid present in the solution, functional parameters (renal blood flow, intrarenal resistance, urinary flow, pH) and oxygenation (arterial pO2, absence of hypoxia-inducible factor 1-alpha) were similar to the pRBC group. Furthermore, taurine significantly improved the antioxidant capacity in the Ecosol group, reflected in decreased lactate dehydrogenase, urine protein and tubular vacuolization compared to pRBC. This study demonstrates the feasibility of 6 h NMP using a taurine containing, cell- and oxygen carrier-free perfusate, achieving a comparable organ quality to pRBC perfused porcine kidneys. [ABSTRACT FROM AUTHOR]

Ischemia-reperfusion injury remains a fundamental problem during organ transplantation logistics. One key technical factor is the rapid allograft rewarming during the time of vascular reconstruction in the recipient. In this pilot study, a new thermal insulation bag (TIB) for organ transplantation was used. Insulation capacity, tissue compatibility, and usability were tested initially ex vivo on porcine kidneys (n = 24) followed by the first in vivo usage. Fourteen female German landrace pigs underwent kidney auto-transplantation after 24 h cold storage (4 °C). During the implantation process the kidney was either insulated with the new TIB, or it was not thermo-protected at all, which represents the clinical standard. In this proof-of-concept study, the usability (knife-to-skin-time) and the general thermal capacity (30 min warm storage at 38 °C ex vivo p < 0.001) was shown. The clinical outcome showed significant differences in the determination of CRP and pi-GST levels. Syndecan-1 Antibody staining showed clear significant higher counts in the control group (p < 0.01) indicating epithelial damage. However, the effect on renal outcomes in not severely pre-damaged kidneys does not appear to be conclusively significant. A close follow-up study is warranted, especially in the context of marginal organs or in cases where anastomosis-times are prolonged due to surgical complexity (e.g., multiple vessels and complex reconstructions). [ABSTRACT FROM AUTHOR]

Objectives: Respiratory failure is the major cause of death in coronavirus disease 2019 (COVID-19). Autopsy-based reports describe diffuse alveolar damage (DAD), organizing pneumonia, and fibrotic change, but data on early pathologic changes and during progression of the disease are rare.Methods: We prospectively enrolled three patients with COVID-19 and performed full clinical evaluation, including high-resolution computed tomography. We took transbronchial biopsy (TBB) specimens at different time points and autopsy tissue samples for histopathologic and ultrastructural evaluation after the patients' death.Results: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed by reverse transcription polymerase chain reaction and/or fluorescence in situ hybridization in all TBBs. Lung histology showed reactive pneumocytes and capillary congestion in one patient who died shortly after hospital admission with detectable virus in one of two lung autopsy samples. SARS-CoV-2 was detected in two of two autopsy samples from another patient with a fulminant course and very short latency between biopsy and autopsy, showing widespread organizing DAD. In a third patient with a prolonged course, autopsy samples showed extensive fibrosis without detectable virus.Conclusions: We report the course of COVID-19 in paired biopsy specimens and autopsies, illustrating vascular, organizing, and fibrotic patterns of COVID-19-induced lung injury. Our results suggest an early spread of SARS-CoV-2 from the upper airways to the lung periphery with diminishing viral load during disease. [ABSTRACT FROM AUTHOR]

Background and Purpose: Macrophage migration inhibitory factor (MIF) is an inflammatory and chemokine-like protein expressed in different inflammatory diseases as well as solid tumours. CD74-as the cognate MIF receptor-was identified as an important target of MIF. We here analysed the role of MIF and CD74 in the progression of hepatocellular carcinoma (HCC) in vitro and in vivo.Experimental Approach: Multilocular HCC was induced using the diethylnitrosamine/carbon tetrachloride (DEN/CCl4 ) model in hepatocyte-specific Mif knockout (Mif Δhep ), Cd74-deficient, and control mice. Tumour burden was compared between the genotypes. MIF, CD74 and Ki67 expression were investigated in tumour and surrounding tissue. In vitro, the effects of the MIF/CD74 axis on the proliferative and apoptotic behaviour of hepatoma cells and respective signalling pathways were assessed after treatment with MIF and anti-CD74 antibodies.Key Results: DEN/CCl4 treatment of Mif Δhep mice resulted in reduced tumour burden and diminished proliferation capacity within tumour tissue. In vitro, MIF stimulated proliferation of Hepa 1-6 and HepG2 cells, inhibited therapy-induced cell death and induced ERK activation. The investigated effects could be reversed using a neutralizing anti-CD74 antibody, and Cd74-/- mice developed fewer tumours associated with decreased proliferation rates.Conclusion and Implications: We identified a pro-tumorigenic role of MIF during proliferation and therapy-induced apoptosis of HCC cells. These effects were mediated via the MIF cognate receptor CD74. Thus, inhibition of the MIF/CD74 axis could represent a promising target with regard to new pharmacological therapies aimed at HCC. [ABSTRACT FROM AUTHOR]

Summary: Virus detection methods are important to cope with the SARS‐CoV‐2 pandemics. Apart from the lung, SARS‐CoV‐2 was detected in multiple organs in severe cases. Less is known on organ tropism in patients developing mild or no symptoms, and some of such patients might be missed in symptom‐indicated swab testing. Here, we tested and validated several approaches and selected the most reliable RT‐PCR protocol for the detection of SARS‐CoV‐2 RNA in patients' routine diagnostic formalin‐fixed and paraffin‐embedded (FFPE) specimens available in pathology, to assess (i) organ tropism in samples from COVID‐19‐positive patients, (ii) unrecognized cases in selected tissues from negative or not‐tested patients during a pandemic peak, and (iii) retrospectively, pre‐pandemic lung samples. We identified SARS‐CoV‐2 RNA in seven samples from confirmed COVID‐19 patients, in two gastric biopsies, one small bowel and one colon resection, one lung biopsy, one pleural resection and one pleural effusion specimen, while all other specimens were negative. In the pandemic peak cohort, we identified one previously unrecognized COVID‐19 case in tonsillectomy samples. All pre‐pandemic lung samples were negative. In conclusion, SARS‐CoV‐2 RNA detection in FFPE pathology specimens can potentially improve surveillance of COVID‐19, allow retrospective studies, and advance our understanding of SARS‐CoV‐2 organ tropism and effects. [ABSTRACT FROM AUTHOR]

Abstract Background Y-box binding protein-1 (YB-1) is the prototypic member of the cold shock protein family that fulfills numerous cellular functions. In the nucleus YB-1 protein orchestrates transcription of proliferation-related genes, whereas in the cytoplasm it associates with mRNA and directs translation. In human tumor entities, such as breast, lung and prostate cancer, cellular YB-1 expression indicates poor clinical outcome, suggesting that YB-1 is an attractive marker to predict patients' prognosis and, potentially, is suitable to individualize treatment protocols. Given these predictive qualities of YB-1 detection we sought to establish a highly specific monoclonal antibody (Mab) for diagnostic testing and its characterization towards outcome prediction (relapse-free and overall survival). Methods Hybridoma cell generation was carried out with recombinant YB-1 protein as immunogen and Mab characterization was performed using immunoblotting and ELISA with recombinant and tagged YB-1 proteins, as well as immunohistochemistry of healthy and breast cancer specimens. Breast tumor tissue array staining results were analyzed for correlations with receptor expression and outcome parameters. Results YB-1-specific Mab F-E2G5 associates with conformational binding epitopes mapping to two domains within the N-terminal half of the protein and detects nuclear YB-1 protein by immunohistochemistry in paraffin-embedded breast cancer tissues. Prognostic evaluation of Mab F-E2G5 was performed by immunohistochemistry of a human breast cancer tissue microarray comprising 179 invasive breast cancers, 8 ductal carcinoma in situ and 37 normal breast tissue samples. Nuclear YB-1 detection in human breast cancer cells was associated with poor overall survival (p = 0.0046). We observed a close correlation between nuclear YB-1 detection and absence of progesterone receptor expression (p = 0.002), indicating that nuclear YB-1 detection marks a specific subgroup of breast cancer. Likely due to limitation of sample size Cox regression models failed to demonstrate significance for nuclear YB-1 detection as independent prognostic marker. Conclusion Monoclonal YB-1 antibody F-E2G5 should be of great value for prospective studies to validate YB-1 as a novel biomarker suitable to optimize breast cancer treatment.

Kidney fibrosis is characterized by expansion and activation of platelet‐derived growth factor receptor‐β (PDGFR‐β)‐positive mesenchymal cells. To study the consequences of PDGFR‐β activation, we developed a model of primary renal fibrosis using transgenic mice with PDGFR‐β activation specifically in renal mesenchymal cells, driving their pathological proliferation and phenotypic switch toward myofibroblasts. This resulted in progressive mesangioproliferative glomerulonephritis, mesangial sclerosis, and interstitial fibrosis with progressive anemia due to loss of erythropoietin production by fibroblasts. Fibrosis induced secondary tubular epithelial injury at later stages, coinciding with microinflammation, and aggravated the progression of hypertensive and obstructive nephropathy. Inhibition of PDGFR activation reversed fibrosis more effectively in the tubulointerstitium compared to glomeruli. Gene expression signatures in mice with PDGFR‐β activation resembled those found in patients. In conclusion, PDGFR‐β activation alone is sufficient to induce progressive renal fibrosis and failure, mimicking key aspects of chronic kidney disease in humans. Our data provide direct proof that fibrosis per se can drive chronic organ damage and establish a model of primary fibrosis allowing specific studies targeting fibrosis progression and regression. Synopsis: Kidney fibrosis is the common pathological process of chronic kidney diseases (CKD), a major global health problem. Using a newly developed murine model of primary fibrosis and analyses of patient samples, this study demonstrated the important role of PDGFR‐β signaling in driving renal fibrosis. Increased mesenchymal expression and activation of PDGFR‐β is a hallmark of both human and animal kidney fibrosis.Expression of constitutively active PDGFR‐β specifically in kidney mesenchymal cells induced their proliferation and activation leading to progressive fibrosis.This model of primary fibrosis led to secondary tubular injury and inflammation at later stages, proving the detrimental renal effects of ongoing fibrosis.Intervention with imatinib, a tyrosine kinase blocker including activated PDGFR, showed differential reversibility of kidney fibrosis in interstitium and glomeruli.Expression profiles in this model resembled those found in patients with kidney fibrosis, supporting the relevance of PDGFR‐β activation. [ABSTRACT FROM AUTHOR]

Here, we show that inducible overexpression of Cre recombinase in glomerular podocytes but not in parietal epithelial cells may trigger focal segmental glomerulosclerosis (FSGS) in juvenile transgenic homocygous Pod-rtTA/LC1 mice. Administration of doxycycline shortly after birth, but not at any other time point later in life, resulted in podocyte injury and development of classical FSGS lesions in these mice. Sclerotic lesions were formed as soon as 3 wk of age, and FSGS progressed with low variability until 13 wk of age. In addition, our experiments identified Cre toxicity as a potentially relevant limitation for studies in podocytes of transgenic animals. In summary, our study establishes a novel genetic model for FSGS in mice, which exhibits low variability and manifests already at a young age. [ABSTRACT FROM AUTHOR]

Abstract Renal fibrosis is the final pathological process common to any ongoing, chronic kidney injury or maladaptive repair. It is considered as the underlying pathological process of chronic kidney disease (CKD), which affects more than 10% of world population and for which treatment options are limited. Renal fibrosis is defined by excessive deposition of extracellular matrix, which disrupts and replaces the functional parenchyma that leads to organ failure. Kidney's histological structure can be divided into three main compartments, all of which can be affected by fibrosis, specifically termed glomerulosclerosis in glomeruli, interstitial fibrosis in tubulointerstitium and arteriosclerosis and perivascular fibrosis in vasculature. In this review, we summarized the different appearance, cellular origin and major emerging processes and mediators of fibrosis in each compartment. We also depicted and discussed the challenges in translation of anti-fibrotic treatment to clinical practice and discuss possible solutions and future directions. [ABSTRACT FROM AUTHOR]

Clinical data after cardiac surgery and experimental mouse models of acute kidney injury suggest a renoprotective role of macrophage migration inhibitory factor (MIF). MIF muffles kidney injury: Patients undergoing open-heart surgery are susceptible to complications, including acute kidney injury (AKI). Stoppe et al. observed that patients who had high serum concentrations of macrophage migration inhibitory factor (MIF) after cardiac surgery had lower risk of AKI. Renal ischemia-reperfusion injury induced more inflammation and kidney cell death in mice lacking Mif than in wild-type mice. Treating isolated kidney cells with MIF protected against hypoxia-induced cell death. Mice treated with MIF or a soluble form of CD74 (MIF receptor) showed reduced kidney injury after ischemia-reperfusion. This study suggests that MIF may protect the kidney from ischemia-reperfusion injury. Acute kidney injury (AKI) represents the most frequent complication after cardiac surgery. Macrophage migration inhibitory factor (MIF) is a stress-regulating cytokine that was shown to protect the heart from myocardial ischemia-reperfusion injury, but its role in the pathogenesis of AKI remains unknown. In an observational study, serum and urinary MIF was quantified in 60 patients scheduled for elective conventional cardiac surgery with the use of cardiopulmonary bypass. Cardiac surgery triggered an increase in MIF serum concentrations, and patients with high circulating MIF (>median) 12 hours after surgery had a significantly reduced risk of developing AKI (relative risk reduction, 72.7%; 95% confidence interval, 12 to 91.5%; P = 0.03). Experimental AKI was induced in wild-type and Mif−/− mice by 30 min of ischemia followed by 6 or 24 hours of reperfusion, or by rhabdomyolysis. Mif-deficient mice exhibited increased tubular cell injury, increased regulated cell death (necroptosis and ferroptosis), and enhanced oxidative stress. Therapeutic administration of recombinant MIF after ischemia-reperfusion in mice ameliorated AKI. In vitro treatment of tubular epithelial cells with recombinant MIF reduced cell death and oxidative stress as measured by glutathione and thiobarbituric acid reactive substances in the setting of hypoxia. Our data provide evidence of a renoprotective role of MIF in experimental ischemia-reperfusion injury by protecting renal tubular epithelial cells, consistent with our observation that high MIF in cardiac surgery patients is associated with a reduced incidence of AKI. [ABSTRACT FROM AUTHOR]

Background: Polycystic kidney disease (PKD) is a genetic disorder affecting millions of people worldwide that is characterized by fluid-filled cysts and leads to end-stage renal disease (ESRD). The hallmarks of PKD are proliferation and dedifferentiation of tubular epithelial cells, cellular processes known to be regulated by Notch signaling. Methods: We found increased Notch3 expression in human PKD and renal cell carcinoma biopsies. To obtain insight into the underlying mechanisms and the functional consequences of this abnormal expression, we developed a transgenic mouse model with conditional overexpression of the intracellular Notch3 (ICN3) domain specifically in renal tubules. We evaluated the alterations in renal function (creatininemia, BUN) and structure (cysts, fibrosis, inflammation) and measured the expression of several genes involved in Notch signaling and the mechanisms of inflammation, proliferation, dedifferentiation, fibrosis, injury, apoptosis and regeneration. Results: After one month of ICN3 overexpression, kidneys were larger with tubules grossly enlarged in diameter, with cell hypertrophy and hyperplasia, exclusively in the outer stripe of the outer medulla. After three months, mice developed numerous cysts in proximal and distal tubules. The cysts had variable sizes and were lined with a single- or multilayered, flattened, cuboid or columnar epithelium. This resulted in epithelial hyperplasia, which was observed as protrusions into the cystic lumen in some of the renal cysts. The pre-cystic and cystic epithelium showed increased expression of cytoskeletal filaments and markers of epithelial injury and dedifferentiation. Additionally, the epithelium showed increased proliferation with an aberrant orientation of the mitotic spindle. These phenotypic tubular alterations led to progressive interstitial inflammation and fibrosis. Conclusions: In summary, Notch3 signaling promoted tubular cell proliferation, the alignment of cell division, dedifferentiation and hyperplasia, leading to cystic kidney diseases and pre-neoplastic lesions. [ABSTRACT FROM AUTHOR]

Background: The Y-box protein-1 (YB-1) fulfills pleiotropic functions relating to gene transcription, mRNA processing, and translation. It remains elusive how YB-1 shuttling into the nuclear and cytoplasmic compartments is regulated and whether limited proteolysis by the 20S proteasome releases fragments with distinct function(s) and subcellular distribution(s). Results: To address these questions, mapping of domains responsible for subcellular targeting was performed. Three nuclear localization signals (NLS) were identified. NLS-1 (aa 149-156) and NLS-2 (aa 185-194) correspond to residues with unknown function(s), whereas NLS-3 (aa 276-292) matches with a designated multimerization domain. Nuclear export signal(s) were not identified. Endoproteolytic processing by the 20S proteasome before glycine 220 releases a carboxy-terminal fragment (CTF), which localized to the nucleus, indicating that NLS-3 is operative. Genotoxic stress induced proteolytic cleavage and nuclear translocation of the CTF. Co-expression of the CTF and full-length YB-1 resulted in an abrogated transcriptional activation of the MMP-2 promoter, indicating an autoregulatory inhibitory loop, whereas it fulfilled similar trans-repressive effects on the collagen type I promoter. Conclusion: Compartmentalization of YB-1 protein derivatives is controlled by distinct NLS, one of which targets a proteolytic cleavage product to the nucleus. We propose a model for an autoregulatory negative feedback loop that halts unlimited transcriptional activation. [ABSTRACT FROM AUTHOR]

The Notch3 receptor participates in the development and maturation of vessels. Mutations of Notch3 in humans are associated with defective regulation of cerebral blood flow. To investigate the role of Notch3 in the regulation of renal hemodynamics, we used mice lacking expression of the Notch3 gene (Notch3-/- mice). Bolus injections of norepinephrine and angiotensin II increased renal vascular resistance and decreased renal blood flow in a dose-dependent manner in wild-type mice. In sharp contrast, renal vascular resistance of Notch3-/- mice varied little after boluses of norepinephrine and angiotensin II. Inversely, bradykinin and prostacyclin relaxed renal vasculature in wild-type mice. Both vasodilators had a negligible effect on renal vascular resistance of Notch3-/- mice. Afferent arterioles freshly isolated from Notch3-/- mice displayed decreased thickness of vascular wall compared with wild -type mice and showed a deficient contractile response to angiotensin II. To examine the physiopathological consequences of the above-described deficiency, hypertension was induced by continuous infusion of angiotensin II. Angiotensin II gradually increased blood pressure in both strains, but this increase was lesser in the Notch3-/- mice. Despite this blunted systemic effect, Notch3-/- mice displayed high mortality rates (65%) attributed to heart failure. In the kidney, the surviving Notch3-/- mice showed focal structural alterations characteristic of nephroangiosclerosis. These data show that Notch3 is necessary for the adaptive response of the renal vasculature to vasoactive systems. A deficiency in the expression of Notch3 could have important physiopathological consequences in the adaptation of the cardiac and renal function to chronic increase of blood pressure. [ABSTRACT FROM AUTHOR]

Y-box protein (YB)-1 of the cold-shock protein family functions in gene transcription and RNA processing. Extracellular functions have not been reported, but the YB-1 staining pattern in inflammatory glomerular diseases, without adherence to cell boundaries, suggests an extracellular occurrence. Here, we show the secretion of YB-1 by mesangial and monocytic cells after inflammatory challenges. It should be noted that YB-1 was secreted through a non-classical mode resembling that of the macrophage migration inhibitory factor. YB-1 release requires ATP-binding cassette transporters, and microvesicles protect YB-1 from protease degradation. Two lysine residues in the YB-1 carboxy-terminal domain are crucial for its release, probably because of post-translational modifications. The addition of purified recombinant YB-1 protein to different cell types results in increased DNA synthesis, cell proliferation and migration. Thus, the non-classically secreted YB-1 has extracellular functions and exerts mitogenic as well as promigratory effects in inflammation. [ABSTRACT FROM AUTHOR]

Multiorgan tropism of SARS-CoV-2 has previously been shown for several major organs. We have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n = 8), using histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA in situ hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. SARS-CoV-2 RNA was mainly localized in epithelial cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. These findings were validated using in situ hybridization on external COVID-19 autopsy samples (n = 9). Apart from the lung, correlation of viral detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2. In summary, SARS-CoV-2 and its replication could be observed across all organ systems, which co-localizes with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine with anti-fibrotic properties in toxic liver injury models and anti-steatotic functions in non-alcoholic fatty liver disease (NAFLD) attributed to the CD74/AMPK signaling pathway. As NAFLD progression is associated with fibrosis, we studied MIF function during NAFLD-associated liver fibrogenesis in mice and men by molecular, histological and immunological methods in vitro and in vivo. After NASH diet feeding, hepatic Mif expression was strongly induced, an effect which was absent in Mif∆hep mice. In contrast to hepatotoxic fibrosis models, NASH diet-induced fibrogenesis was significantly abrogated in Mif−/− and Mif∆hep mice associated with a reduced accumulation of the pro-fibrotic type-I NKT cell subpopulation. In vitro, MIF skewed the differentiation of NKT cells towards the type-I subtype. In line with the murine results, expression of fibrosis markers strongly correlated with MIF, its receptors, and markers of NKT type-I cells in NASH patients. We conclude that MIF expression is induced during chronic metabolic injury in mice and men with hepatocytes representing the major source. In NAFLD progression, MIF contributes to liver fibrogenesis skewing NKT cell polarization toward a pro-fibrotic phenotype highlighting the complex, context-dependent role of MIF during chronic liver injury. [ABSTRACT FROM AUTHOR]

Elastin-specific MRI allows quantitative and longitudinal renal fibrosis assessment and monitoring of treatment efficacy. Finding fibrosis: Excessive extracellular matrix deposition in the kidney impairs renal function, contributing to chronic kidney disease. As an alternative to needle-based biopsies used to diagnose and stage kidney disease, Sun et al. used an elastin-specific imaging agent, ESMA, to noninvasively analyze kidney fibrosis. Elastin was overexpressed in renal tissue from patients with kidney fibrosis, and ESMA identified fibrotic areas in human kidneys ex vivo. Similar results were seen in vivo using three mouse models of kidney fibrosis. ESMA could be used to track the progression of renal fibrosis and response to imatinib therapy in mice. These results support the use of ESMA-based imaging for noninvasive renal fibrosis monitoring. Fibrosis is the common endpoint and currently the best predictor of progression of chronic kidney diseases (CKDs). Despite several drawbacks, biopsies remain the only available means to specifically assess the extent of renal fibrosis. Here, we show that molecular imaging of the extracellular matrix protein elastin allows for noninvasive staging and longitudinal monitoring of renal fibrosis. Elastin was hardly expressed in healthy mouse, rat, and human kidneys, whereas it was highly up-regulated in cortical, medullar, and perivascular regions in progressive CKD. Compared to a clinically relevant control contrast agent, the elastin-specific magnetic resonance imaging agent ESMA specifically detected elastin expression in multiple mouse models of renal fibrosis and also in fibrotic human kidneys. Elastin imaging allowed for repetitive and reproducible assessment of renal fibrosis, and it enabled longitudinal monitoring of therapeutic interventions, accurately capturing anti-fibrotic therapy effects. Last, in a model of reversible renal injury, elastin imaging detected ensuing fibrosis not identifiable via routine assessment of kidney function. Elastin imaging thus has the potential to become a noninvasive, specific imaging method to assess renal fibrosis. [ABSTRACT FROM AUTHOR]

Anti-cytokine therapies have substantially improved the treatment of inflammatory and autoimmune diseases. Cytokine-targeting drugs are usually biologics such as antibodies or other engineered proteins. Production of biologics, however, is complex and intricate and therefore expensive which might limit therapeutic application. To overcome this limitation we developed a strategy that involves the design of an optimized, monogenic cytokine inhibitor and the protein producing capacity of the host. Here, we engineered and characterized a receptor fusion protein, mIL-6-RFP-Fc, for the inhibition of interleukin-6 (IL-6), a well-established target in anti-cytokine therapy. Upon application in mice mIL-6-RFP-Fc inhibited IL-6-induced activation of the transcription factor STAT3 and ERK1/2 kinases in liver and kidney. mIL-6-RFP-Fc is encoded by a single gene and therefore most relevant for gene transfer approaches. Gene transfer through hydrodynamic plasmid delivery in mice resulted in hepatic production and secretion of mIL-6-RFP-Fc into the blood in considerable amounts, blocked hepatic acute phase protein synthesis and improved kidney function in an ischemia and reperfusion injury model. Our study establishes receptor fusion proteins as promising agents in anti-cytokine therapies through gene therapeutic approaches for future targeted and cost-effective treatments. The strategy described here is applicable for many cytokines involved in inflammatory and other diseases. [ABSTRACT FROM AUTHOR]

Background: Y-box binding protein-1 (YB-1) is the prototypic member of the cold shock protein family that fulfills numerous cellular functions. In the nucleus YB-1 protein orchestrates transcription of proliferation-related genes, whereas in the cytoplasm it associates with mRNA and directs translation. In human tumor entities, such as breast, lung and prostate cancer, cellular YB-1 expression indicates poor clinical outcome, suggesting that YB-1 is an attractive marker to predict patients' prognosis and, potentially, is suitable to individualize treatment protocols. Given these predictive qualities of YB-1 detection we sought to establish a highly specific monoclonal antibody (Mab) for diagnostic testing and its characterization towards outcome prediction (relapse-free and overall survival).Methods: Hybridoma cell generation was carried out with recombinant YB-1 protein as immunogen and Mab characterization was performed using immunoblotting and ELISA with recombinant and tagged YB-1 proteins, as well as immunohistochemistry of healthy and breast cancer specimens. Breast tumor tissue array staining results were analyzed for correlations with receptor expression and outcome parameters.Results: YB-1-specific Mab F-E2G5 associates with conformational binding epitopes mapping to two domains within the N-terminal half of the protein and detects nuclear YB-1 protein by immunohistochemistry in paraffin-embedded breast cancer tissues. Prognostic evaluation of Mab F-E2G5 was performed by immunohistochemistry of a human breast cancer tissue microarray comprising 179 invasive breast cancers, 8 ductal carcinoma in situ and 37 normal breast tissue samples. Nuclear YB-1 detection in human breast cancer cells was associated with poor overall survival (p = 0.0046). We observed a close correlation between nuclear YB-1 detection and absence of progesterone receptor expression (p = 0.002), indicating that nuclear YB-1 detection marks a specific subgroup of breast cancer. Likely due to limitation of sample size Cox regression models failed to demonstrate significance for nuclear YB-1 detection as independent prognostic marker.Conclusion: Monoclonal YB-1 antibody F-E2G5 should be of great value for prospective studies to validate YB-1 as a novel biomarker suitable to optimize breast cancer treatment. [ABSTRACT FROM AUTHOR]

In vitro studies identified Y-box-binding protein (YB)-1 as a key regulator of inflammatory mediators. In this study, we observed increased levels of secreted YB-1 in sera from sepsis patients. This led us to investigate the in vivo role of YB-1 in murine models of acute peritonitis following LPS injection, in sterile renal inflammation following unilateral ureteral obstruction, and in experimental pyelonephritis. LPS injection enhanced de novo secretion of YB-1 into the urine and the peritoneal fluid of LPS-treated mice. Furthermore, we could demonstrate a significant, transient upregulation and posttranslational modification (phosphorylation at serine 102) of YB-1 in renal and inflammatory cells. Increased renal cytoplasmic YB-1 amounts conferred enhanced expression of proinflammatory chemokines CCL2 and CCL5. Along these lines, heterozygous YB-1 knockout mice (YB-1+/d) that display 50% reduced YB-1 levels developed significantly lower responses to both LPS and sterile inflammation induced by unilateral ureteral obstruction. This included diminished immune cell numbers due to impaired migration propensities and reduced chemokine expression. YB-1+/d mice were protected from LPS-associated mortality (20% mortality on day 3 versus 80% in wild-type controls); however, immunosuppression in YB-1+/d animals resulted in 50% mortality. In conclusion, our findings identify YB-1 as a major, nonredundant mediator in both systemic and local inflammatory responses. [ABSTRACT FROM AUTHOR]

Abstract: The cold shock protein Y-box (YB) binding-1 is an example of a highly regulated protein with pleiotropic functions. Besides activities as a transcription factor in the nucleus or regulator of translation in the cytoplasm, recent findings indicate extracellular effects and secretion via a non-classical secretion pathway. This review summarizes regulatory pathways in which YB-1 participates, all iterating auto-regulatory loops. Schematics are developed that elucidate the cold shock protein activities in (i) fine-tuning its own expression level following platelet-derived growth factor-B-, thrombin- or interferon-γ-dependent signaling, (ii) as a component of the messenger ribonucleoprotein (mRNP) complex for interleukin-2 synthesis in T-cell commitment/activation, (iii) pro-fibrogenic cell phenotypic changes mediated by transforming growth factor-β, and (iv) receptor Notch-3 cleavage and signal transduction. Emphasis is put forward on subcellular protein translocation mechanisms and underlying signaling pathways. These have mostly been analysed in cell culture systems and rarely in experimental models. In sum, YB-1 seems to fulfill a pacemaker role in diverse diseases, both inflammatory/pro-fibrogenic as well as tumorigenic. A clue towards potential intervention strategies may reside in the understanding of the outlined auto-regulatory loops and means to interfere with cycling pathways. [Copyright &y& Elsevier]

The Y-box protein-1 (YB-1) belongs to the family of cold shock proteins that have pleiotropic functions such as gene transcription, RNA splicing, and mRNA translation. YB-1 has a critical role in atherogenesis due to its regulatory effects on chemokine CCL5 (RANTES) gene transcription in vascular smooth muscle cells. Since CCL5 is a key mediator of kidney transplant rejection, we determined whether YB-1 is involved in allograft rejection by manipulating its expression. In human kidney biopsies, YB-1 transcripts were amplified 17-fold in acute and 21-fold in chronic allograft rejection with a close correlation between CCL5 and YB-1 mRNA expression in both conditions. Among three possible YB-1 binding sites in the CCL5 promoter, a critical element was mapped at −28/−10 bps. This site allowed up-regulation of CCL5 transcription in monocytic THP-1 and HUT78 T-cells and in human primary monocytes; however, it repressed transcription in differentiated macrophages. Conversely, YB-1 knockdown led to decreased CCL5 transcription and secretion in monocytic cells. We show that YB-1 is a cell-type specific regulator of CCL5 expression in infiltrating T-cells and monocytes/macrophages and acts as an adaptive controller of inflammation during kidney allograft rejection.Kidney International (2009) 75, 185–196; doi:10.1038/ki.2008.457; published online 17 September 2008 [ABSTRACT FROM AUTHOR]

Several studies revealed alterations of single sphingolipid species, such as chain length-specific ceramides, in plasma and serum of patients with kidney diseases. Here, we investigated whether such alterations occur in kidney tissue from patients and mice suffering from renal fibrosis, the common endpoint of chronic kidney diseases. Human fibrotic kidney samples were collected from nephrectomy specimens with hydronephrosis and/or pyelonephritis. Healthy parts from tumor nephrectomies served as nonfibrotic controls. Mouse fibrotic kidney samples were collected from male C57BL/6J mice treated with an adenine-rich diet for 14 days or were subjected to 7 days of unilateral ureteral obstruction (UUO). Kidneys of untreated mice and contralateral kidneys (UUO) served as respective controls. Sphingolipid levels were detected by LC-MS/MS. Fibrotic markers were analyzed by TaqMan® analysis and immunohistology. Very long-chain ceramides Cer d18:1/24:0 and Cer d18:1/24:1 were significantly downregulated in both fibrotic human kidney cortex and fibrotic murine kidney compared to respective control samples. These effects correlate with upregulation of COL1α1, COL3α1 and αSMA expression in fibrotic human kidney cortex and fibrotic mouse kidney. We have shown that very long-chain ceramides Cer d18:1/24:0 and Cer d18:1/24:1 are consistently downregulated in fibrotic kidney samples from human and mouse. Our findings support the use of in vivo murine models as appropriate translational means to understand the involvement of ceramides in human kidney diseases. In addition, our study raises interesting questions about the possible manipulation of ceramide metabolism to prevent progression of fibrosis and the use of ceramides as potential biomarkers of chronic kidney disease. • Very long-chain ceramides are downregulated in fibrotic renal tissue. • Regulation of ceramides in fibrotic kidneys is similar in mice and men. • Different ceramide profile in human renal cortex compared to renal medulla [ABSTRACT FROM AUTHOR]

Abstract Macrophage migration inhibitory factor (MIF) is a cytokine expressed in various cell types, including hematopoietic, epithelial, endothelial, mesenchymal and neuronal cells. Altered MIF expression has been associated with a multitude of diseases ranging from inflammatory disorders like sepsis, lupus and rheumatoid arthritis to organ pathologies such as heart failure, myocardial infarction, acute kidney injury, organ fibrosis and a number of malignancies. The implication of MIF in these diseases was supported by numerous animal studies. MIF acts in an autocrine and paracrine manner via binding and activating the receptors CD74/CD44, CXCR2, CXCR4 and CXCR7. Upon receptor binding, several downstream signaling pathways were shown to be activated in vivo, including ERK1/2, AMPK and AKT. Expression of MIF receptors is not uniform in various cells, resulting in differential responses to MIF across various tissues and pathologies. Within cells, MIF can directly bind and interact with intracellular proteins, such as the constitutive photomorphogenic-9 (COP9) signalosome subunit 5 (CSN5), p53 or thioredoxin-interacting protein (TXNIP). D-dopachrome tautomerase (D-DT or MIF-2) was recognized to be a structural and functional homolog of MIF, which could exert overlapping effects, raising further the complexity of canonical MIF signaling pathways. Here, we provide an overview of the expression and regulation of MIF, D-DT and their receptors. We also discuss the downstream signaling pathways regulated by MIF/D-DT and their pathological roles in different tissue, particularly in the heart and the kidney. [ABSTRACT FROM AUTHOR]

Background & Aims Non-alcoholic fatty liver (NAFL) is the hepatic consequence of metabolic syndrome and can progress to non-alcoholic steatohepatitis (NASH). The identification of molecular and cellular factors that determine the progression of NASH and lead to irreversible hepatocellular damage are crucial. Dendritic cells (DCs) represent a heterogeneous cell population among which CD103 + DCs play a significant role in immunity and tolerance. We aimed to clarify the role of this DC subset in the pathomechanism of NASH. Methods Steatosis progression towards steatohepatitis was analysed using multicolor FACS analyses, cytokine and qPCR array in high sucrose diet (HSD) and methionine and choline deficient diet (MCD) fed wild-type and basic leucine zipper transcription factor, ATF-Like-3 ( Batf3 ) deficient animals, which lack CD103 + DCs (classical type-1 DC, cDC1s). Results Metabolic challenge of Batf3 −/− animals resulted in the progression of steatosis towards steatohepatitis, manifesting by an increased influx of inflammatory cells into the liver and elevated inflammatory cytokine production of myeloid cells upon innate stimuli. However, the lack of cDC1s did not affect cellular apoptosis and fibrosis progression but altered genes involved in lipid metabolism. The adoptive transfer of CD103 + cDC1s to Batf3 deficient animals reversed these observed changes and more importantly could attenuate cellular damage and inflammation in established murine steatohepatitis. Conclusion Here, we have identified the murine CD103 + cDC1s as a protective DC subtype that influences the pro–anti-inflammatory balance and protects the liver from metabolic damage. As guardians of liver integrity, they play a key role in the inflammatory process during the development of steatohepatitis in mice. Lay summary Non-alcoholic fatty liver (NAFL) is the hepatic consequence of metabolic syndrome and can lead to non-alcoholic steatohepatitis (NASH). The current study demonstrated that a specific murine dendritic cell subtype possesses a potent regulatory role to influence the inflammatory milieu of the liver in this process. [ABSTRACT FROM AUTHOR]

Crescentic glomerulonephritis (cGN) is the most aggressive form of glomerulonephritis in humans. A widely studied mouse model is induced by sheep or rabbit antisera raised against murine renal cortical antigens. We here, report that Alpaca readily produce ample amounts of antisera that induces pathology in mice, resembling human disease regarding crescent formation, proteinuria, infiltrating immune cells and a significant Th1, but not Th17 immune response. Alpaca antiserum did not cause end-stage kidney failure, neither in a passive nor in an accelerated experimental setting, which may be advantageous for long term studies of crescentic glomerulonephritis. • Alpacas produce ample amounts of antisera that induces pathology resembling human disease regarding crescent formation and proteinuria. • Alpaca antiserum does not cause end-stage kidney failure, neither in a passive nor in an accelerated experimental setting. • These characteristics may be advantageous for long term study of proliferative glomerulonephritis. [ABSTRACT FROM AUTHOR]

The immunosuppressive calcineurin inhibitors (CNIs) cyclosporine A (CsA) and tacrolimus are widely used in transplant organ recipients, but in the kidney allograft, they may cause tubulointerstitial as well as mesangial fibrosis, with TGF-β believed to be a central inductor. In this study, we report that the cold-shock protein Y-box binding protein-1 (YB-1) is a TGF-β independent downstream effector in CsA- as well as in tacrolimus- but not in rapamycin-mediated activation of rat mesangial cells (rMCs). Intracellular content of YB-1 is several-fold increased in MCs following CNI treatment in vitro and in vivo in mice. This effect ensues in a time-dependent manner, and the operative concentration range encompasses therapeutically relevant doses for CNIs. The effect of CNI on cellular YB-1 content is abrogated by specific blockade of translation, whereas retarding the transcription remains ineffective. The activation of rMCs by CNIs is accomplished by generation of reactive oxygen species. In contrast to TGF-β-triggered reactive oxygen species generation, hydrogen peroxide especially could be identified as a potent inductor of YB-1 accumulation. In line with this, hindering TGF-β did not influence CNI-induced YB-1 upregulation, whereas ERK/Akt pathways are involved in CNI-mediated YB-1 expression. CsA-induced YB-1 accumulation results in mRNA stabilization and subsequent generation of collagen. Our results provide strong evidence for a CNI-dependent induction of YB-1 in MCs that contributes to renal fibrosis via regulation of its own and collagen translation. [ABSTRACT FROM AUTHOR]

Y-box (YB) protein-1 is secreted by mesangial and immune cells after cytokine challenge, but extracellular functions are unknown. Here, we demonstrate that extracellular YB-1 associates with outer cell membrane components and interacts with extracellular Notch-3 receptor domains. The interaction appears to be specific for Notch-3, as YB-1-green fluorescent protein binds to the extracellular domains and full-length forms of Notch-3 but not to Notch-1. YB-1-green fluorescent protein and Notch-3 proteins co-localize at cell membranes, and extracellular YB-1 activates Notch-3 signaling, resulting in nuclear translocation of the Notch-3 intracellular domain and up-regulation of Notch target genes. The YB-1/Notch-3 interaction may be of particular relevance for inflammatory mesangioproliferative disease, as both proteins co-localize in an experimental nephritis model and receptor activation temporally and spatially correlates with YB-1 expression. [ABSTRACT FROM AUTHOR]

COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS. [Display omitted] • Monocyte-derived macrophages accumulate in the lung in COVID-19 ARDS • Macrophages in COVID-19 express genes associated with profibrotic functions • Patients with severe COVID-19 ARDS display hallmarks of pulmonary fibrosis • SARS-CoV-2 induces a profibrotic transcriptome and proteome profile in macrophages SARS-CoV-2 infection, but not influenza A, triggers immunological and pathological changes in the lung that are hallmarks of pulmonary fibrosis. A subset of CD163+ macrophages are found to drive this fibroproliferative acute respiratory distress. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) leads to a gradual loss of kidney function, with fibrosis as pathological endpoint, which is characterized by extracellular matrix (ECM) deposition and remodeling. Traditionally, in vivo models are used to study interstitial fibrosis, through histological characterization of biopsy tissue. However, ethical considerations and the 3Rs (replacement, reduction, and refinement) regulations emphasizes the need for humanized 3D in vitro models. This study introduces a bioprinted in vitro model which combines primary human cells and decellularized and partially digested extracellular matrix (ddECM). A protocol was established to decellularize kidney pig tissue and the ddECM was used to encapsulate human renal cells. To investigate fibrosis progression, cells were treated with transforming growth factor beta 1 (TGF-β1), and the mechanical properties of the ddECM hydrogel were modulated using vitamin B2 crosslinking. The bioprinting perfusable model replicates the renal tubulointerstitium. Results show an increased Young's modulus over time, together with the increase of ECM components and cell dedifferentiation toward myofibroblasts. Multiple fibrotic genes resulted upregulated, and the model closely resembled fibrotic human tissue in terms of collagen deposition. This 3D bioprinted model offers a more physiologically relevant platform for studying kidney fibrosis, potentially improving disease progression research and high-throughput drug screening., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human-induced pluripotent stem-cell-derived kidney organoids with SARS-CoV-2. Single-cell RNA sequencing indicated injury and dedifferentiation of infected cells with activation of profibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in long COVID., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)