The role of desmoglein-2 in kidney disease.

Desmosomes are multi-protein cell-cell adhesion structures supporting cell stability and mechanical stress resilience of tissues, best described in skin and heart. The kidney is exposed to various mechanical stimuli and stress, yet little is known about kidney desmosomes. In healthy kidneys, we found desmosomal proteins located at the apical-junctional complex in tubular epithelial cells. In four different animal models and patient biopsies with various kidney diseases, desmosomal components were significantly upregulated and partly miss-localized outside of the apical-junctional complexes along the whole lateral tubular epithelial cell membrane. The most upregulated component was desmoglein-2 (Dsg2). Mice with constitutive tubular epithelial cell-specific deletion of Dsg2 developed normally, and other desmosomal components were not altered in these mice. When challenged with different types of tubular epithelial cell injury (unilateral ureteral obstruction, ischemia-reperfusion, and 2,8-dihydroxyadenine crystal nephropathy), we found increased tubular epithelial cell apoptosis, proliferation, tubular atrophy, and inflammation compared to wild-type mice in all models and time points. In vitro, silencing DSG2 via siRNA weakened cell-cell adhesion in HK-2 cells and increased cell death. Thus, our data show a prominent upregulation of desmosomal components in tubular cells across species and diseases and suggest a protective role of Dsg2 against various injurious stimuli.

Keratin Expression in Podocytopathies, ANCA-Associated Vasculitis and IgA Nephropathy.

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.

3D Printed Tubulointerstitium Chip as an In Vitro Testing Platform.

Chronic kidney disease (CKD) ranks as the twelfth leading cause of death worldwide with limited treatment options. The development of in vitro models replicating defined segments of the kidney functional units, the nephrons, in a physiologically relevant and reproducible manner can facilitate drug testing. The aim of this study was to produce an in vitro organ-on-a-chip platform with extrusion-based three-dimensional (3D) printing. The manufacturing of the tubular platform was produced by printing sacrificial fibers with varying diameters, providing a suitable structure for cell adhesion and proliferation. The chip platform was seeded with primary murine tubular epithelial cells and human umbilical vein endothelial cells. The effect of channel geometry, its reproducibility, coatings for cell adhesion, and specific cell markers were investigated. The developed chip presents single and dual channels, mimicking segments of a renal tubule and the capillary network, together with an extracellular matrix gel analogue placed in the middle of the two channels, envisioning the renal tubulointerstitium in vitro. The 3D printed platform enables perfusable circular cross-section channels with fully automated, rapid, and reproducible manufacturing processes at low costs. This kidney tubulointerstitium on-a-chip provides the first step toward the production of more complex in vitro models for drug testing.

Down-regulation of human long non-coding RNA LINC01187 is associated with nephropathies.

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.

Taurine as Antioxidant in a Novel Cell- and Oxygen Carrier-Free Perfusate for Normothermic Machine Perfusion of Porcine Kidneys.

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.

A Proof-of-Concept Preclinical Study Using a Novel Thermal Insulation Device in a Porcine Kidney Auto-Transplantation Model.

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).

Generation of an alpaca serum that induces immune-mediated crescentic glomerulonephritis in mice.

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.

Current kidney function parameters overestimate kidney tissue repair in reversible experimental kidney disease.

Although underlying mechanisms and the clinical course of kidney disease progression are well described, less is known about potential disease reversibility. Therefore, to analyze kidney recovery, we adapted a commonly used murine chronic kidney disease (CKD) model of 2,8- dihydroxyadenine (2,8-DHA) crystal-induced nephropathy to study disease recovery and efficacy of disease-modifying interventions. The recovery phase after CKD was characterized by improved kidney function after two weeks which remained stable thereafter. By contrast, even after eight weeks recovery, tubular injury and inflammation were only partially reduced, and fibrosis persisted. Deep-learning-based histologic analysis of 8,604 glomeruli and 596,614 tubular cross sections revealed numerous tubules had undergone either prominent dilation or complete atrophy, leading to atubular glomeruli and irreversible nephron loss. We confirmed these findings in a second CKD model, reversible unilateral ureteral obstruction, in which a rapid improvement of glomerular filtration rate during recovery also did not reflect the permanent histologic kidney injury. In 2,8-DHA nephropathy, increased drinking volume was highly effective in disease prevention. However, in therapeutic approaches, high fluid intake was only effective in moderate but not severe CKD and established tissue injury was again poorly reflective of kidney function parameters. The injury was particularly localized in the medulla, which is often not analyzed. Thus, recovery after crystal- or obstruction-induced CKD is characterized by ongoing tissue injury, fibrosis, and nephron loss, but not reflected by standard measures of kidney function. Hence, our data might aid in designing kidney recovery studies and suggest the need for biomarkers specifically monitoring intra-kidney tissue injury.

Activation of Notch3 in Renal Tubular Cells Leads to Progressive Cystic Kidney Disease.

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.

Complement activation induces excessive T cell cytotoxicity in severe COVID-19.

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. Proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids.

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.

Antemortem vs Postmortem Histopathologic and Ultrastructural Findings in Paired Transbronchial Biopsy Specimens and Lung Autopsy Samples From Three Patients With Confirmed SARS-CoV-2.

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.

Deep learning-based classification of kidney transplant pathology: a retrospective, multicentre, proof-of-concept study.

BACKGROUND: Histopathological assessment of transplant biopsies is currently the standard method to diagnose allograft rejection and can help guide patient management, but it is one of the most challenging areas of pathology, requiring considerable expertise, time, and effort. We aimed to analyse the utility of deep learning to preclassify histology of kidney allograft biopsies into three main broad categories (ie, normal, rejection, and other diseases) as a potential biopsy triage system focusing on transplant rejection. METHODS: We performed a retrospective, multicentre, proof-of-concept study using 5844 digital whole slide images of kidney allograft biopsies from 1948 patients. Kidney allograft biopsy samples were identified by a database search in the Departments of Pathology of the Amsterdam UMC, Amsterdam, Netherlands (1130 patients) and the University Medical Center Utrecht, Utrecht, Netherlands (717 patients). 101 consecutive kidney transplant biopsies were identified in the archive of the Institute of Pathology, RWTH Aachen University Hospital, Aachen, Germany. Convolutional neural networks (CNNs) were trained to classify allograft biopsies as normal, rejection, or other diseases. Three times cross-validation (1847 patients) and deployment on an external real-world cohort (101 patients) were used for validation. Area under the receiver operating characteristic curve (AUROC) was used as the main performance metric (the primary endpoint to assess CNN performance). FINDINGS: Serial CNNs, first classifying kidney allograft biopsies as normal (AUROC 0·87 [ten times bootstrapped CI 0·85-0·88]) and disease (0·87 [0·86-0·88]), followed by a second CNN classifying biopsies classified as disease into rejection (0·75 [0·73-0·76]) and other diseases (0·75 [0·72-0·77]), showed similar AUROC in cross-validation and deployment on independent real-world data (first CNN normal AUROC 0·83 [0·80-0·85], disease 0·83 [0·73-0·91]; second CNN rejection 0·61 [0·51-0·70], other diseases 0·61 [0·50-0·74]). A single CNN classifying biopsies as normal, rejection, or other diseases showed similar performance in cross-validation (normal AUROC 0·80 [0·73-0·84], rejection 0·76 [0·66-0·80], other diseases 0·50 [0·36-0·57]) and generalised well for normal and rejection classes in the real-world data. Visualisation techniques highlighted rejection-relevant areas of biopsies in the tubulointerstitium. INTERPRETATION: This study showed that deep learning-based classification of transplant biopsies could support pathological diagnostics of kidney allograft rejection. FUNDING: European Research Council; German Research Foundation; German Federal Ministries of Education and Research, Health, and Economic Affairs and Energy; Dutch Kidney Foundation; Human(e) AI Research Priority Area of the University of Amsterdam; and Max-Eder Programme of German Cancer Aid.

SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis.

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.

Multisystemic Cellular Tropism of SARS-CoV-2 in Autopsies of COVID-19 Patients.

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.

Macrophage migration inhibitory factor exerts pro-proliferative and anti-apoptotic effects via CD74 in murine hepatocellular carcinoma.

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.

SARS-CoV-2 RNA screening in routine pathology specimens.

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.

Unexpected Pro-Fibrotic Effect of MIF in Non-Alcoholic Steatohepatitis Is Linked to a Shift in NKT Cell Populations.

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.

Consistent alteration of chain length-specific ceramides in human and mouse fibrotic kidneys.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.