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Purpose: In an effort to reduce waitlist mortality, extended criteria donor organs, including those from donation after circulatory death (DCD), are being used with increasing frequency. These donors carry an increased risk for postoperative complications, and balancing donor-recipient risks is currently based on generalized nomograms. Abdominal normothermic regional perfusion (aNRP) enables individual evaluation of DCD organs, but a gold standard to determine suitability for transplantation is lacking. This study aimed to incorporate individualized and predictive measurements of the liver maximum capacity (LiMAx) test to objectively grade liver function during aNRP and prevent post-op complications., Methods: aNRP was performed to salvage 18 DCD liver grafts, otherwise discarded. Continuous variables were presented as the median with the interquartile range., Results: The liver function maximum capacity (LiMAx) test was successfully performed within the aNRP circuit in 17 aNRPs (94%). Donor livers with good lactate clearance during aNRP demonstrated significantly higher LiMAx scores (396 (301-451) µg/kg/h versus those who did not 105 (70-158) µg/kg/h; P  = 0.006). This was also true for manifesting stress hyperglycemia > 20 mmol/l ( P  = 0.032). LiMAx score correlated with alanine aminotransferase (ALT; R  =  - 0.755) and aspartate transaminase (AST; R  =  - 0.800) levels during perfusion and distinguished livers that were selected for transplantation (397 (346-453) µg/kg/h) from those who were discarded (155 (87-206) µg/kg/h; P  < 0.001). Twelve livers were accepted for transplantation, blinded for LiMAx results, and all had LiMAx scores of > 241 µg/kg/h. Postoperatively, LiMAx during aNRP displayed correlation with 24-h lactate levels., Conclusions: This study shows for the first time the feasibility to assess liver function during aNRP in individual donor livers. LiMAx presents an objective tool to predict donor liver function and risk of complications in the recipient, thus enabling individualized matching of donor livers for an individual recipient. The LiMAx test may present a valuable test for the prediction of donor liver function, preventing post-transplant complication, and personalizing the selection of donor livers for individual recipients., Supplementary Information: The online version contains supplementary material available at 10.1007/s13167-024-00371-7., Competing Interests: Competing interestsThe authors have no conflict of interest., (© The Author(s) 2024.)

Objectives: The human oral microbiome may play a role in the development of oral squamous cell carcinoma. The aim of this scoping review was to examine microbial diversity and differences in the composition of the oral microbiome between OSCC patients and healthy controls., Methods: A literature search (in PubMed and Embase.com) was performed on January 9, 2023. The outcome variables used from the included studies of this review were alpha- and beta diversity and oral microbiome composition profiles for each taxonomic level (phylum-, class-, order-, genus- and species level)., Results: Thirteen out of 423 studies were included in this review compromising 1,677 subjects, of which 905 (54.0%) were OSCC patients and 772 (46.0%) were healthy controls. Most studies found a higher alpha diversity in the OSCC patient group and significantly different beta diversities between OSCC patient samples and healthy control samples. Studies reported more abundant Fusobacteria (on phylum level), Fusobacterium (on genus level), Fusobacterium nucleatum, Porphyromonas endodontalis and Prevotella intermedia (on species level) in OSCC patients. The healthy control group had more abundant Actinobacteria (on phylum level), Streptococcus and Veilonella (on genus level) and Veilonella parvula (on species level) according to most studies., Conclusions: Our findings show differences in oral microbiome diversity and composition in OSCC patients. Clinical implications demand continuing study. Development of internationally accepted standard procedures for oral sample collection and oral microbiota analysis is needed for more conclusive and clinically relevant comparisons in future research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 van Dijk, Petersen, Raber-Durlacher, Epstein and Laheij.)

Iron deficiency anemia can be treated with oral or intravenous Fe supplementation. Such supplementation has considerable effects on the human microbiome, and on opportunistic pathogenic micro-organisms. Molecular understanding of the control and regulation of Fe availability at the host-microbe interface is crucial to interpreting the side effects of Fe supplementation. Here, we provide a concise overview of the regulation of Fe by the opportunistic pathogen Staphylococcus aureus . Ferric uptake regulator (Fur) plays a central role in controlling Fe uptake, utilization and storage in order to maintain a required value. The micro-organism has a strong preference for heme iron as an Fe source, which is enabled by the Iron-regulated surface determinant (Isd) system. The strategies it employs to overcome Fe restriction imposed by the host include: hijacking host proteins, replacing metal cofactors, and replacing functions by non-metal dependent enzymes. We propose that integrated omics approaches, which include metalloproteomics, are necessary to provide a comprehensive understanding of the metal tug of war at the host-microbe interface down to the molecular level., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 van Dijk, de Kruijff and Hagedoorn.)

In renal transplantation, use of calcineurin inhibitors (CNIs) is associated with nephrotoxicity and immunosuppression with malignancies and infections. This trial aimed to minimize CNI exposure and total immunosuppression while maintaining efficacy. We performed a randomized controlled, open-label multicenter trial with early cyclosporine A (CsA) elimination. Patients started with basiliximab, prednisolone (P), mycophenolate sodium (MPS), and CsA. At 6 months, immunosuppression was tapered to P/CsA, P/MPS, or P/everolimus (EVL). Primary outcomes were renal fibrosis and inflammation. Secondary outcomes were estimated glomerular filtration rate (eGFR) and incidence of rejection at 24 months. The P/MPS arm was prematurely halted. The trial continued with P/CsA (N = 89) and P/EVL (N = 96). Interstitial fibrosis and inflammation were significantly decreased and the eGFR was significantly higher in the P/EVL arm. Cumulative rejection rates were 13% (P/EVL) and 19% (P/CsA), (p = 0.08). A post hoc analysis of HLA and donor-specific antibodies at 1 year after transplantation revealed no differences. An individualized immunosuppressive strategy of early CNI elimination to dual therapy with everolimus was associated with decreased allograft fibrosis, preserved allograft function, and good efficacy, but also with more serious adverse events and discontinuation. This can be a valuable alternative regimen in patients suffering from CNI toxicity., (© Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Percutaneous coronary intervention is widely adopted to treat patients with coronary artery disease. However, restenosis remains an unsolved clinical problem after vascular interventions. The role of the systemic and local immune response in the development of restenosis is not fully understood. Hence, the aim of the current study was to investigate the role of the human immune system on subsequent neointima formation elicited by vascular injury in a humanized mouse model. Immunodeficient NOD.Cg-Prkdc scid IL2rg tm1Wjl (NSG) mice were reconstituted with human (h)PBMCs immediately after both carotid wire and femoral cuff injury were induced in order to identify how differences in the severity of injury influenced endothelial regeneration, neointima formation, and homing of human inflammatory and progenitor cells. In contrast to non-reconstituted mice, hPBMC reconstitution reduced neointima formation after femoral cuff injury whereas hPBMCs promoted neointima formation after carotid wire injury 4 weeks after induction of injury. Neointimal endothelium and smooth muscle cells in the injured arteries were of mouse origin. Our results indicate that the immune system may differentially respond to arterial injury depending on the severity of injury, which may also be influenced by the intrinsic properties of the arteries themselves, resulting in either minimal or aggravated neointima formation.

Syndecan-1 is a transmembrane heparan sulfate proteoglycan involved in regenerative growth and cellular adhesion. We hypothesized that the induction of tubular syndecan-1 is a repair response to incipient renal damage in apparently stable, uncomplicated renal transplant recipients. We quantified tubular syndecan-1 in unselected renal protocol biopsies taken 1 yr after transplantation. Spearman rank correlation analysis revealed an inverse correlation between tubular syndecan-1 expression and creatinine clearance at the time of biopsy (r = -0.483, P < 0.03). In a larger panel of protocol and indication biopsies from renal transplant recipients, tubular syndecan-1 correlated with tubular proliferation marker Ki67 (r = 0.518, P < 0.0001). In a rat renal transplantation model, 2 mo after transplantation, mRNA expression of syndecan-1 and its major sheddase, A disintegrin and metalloproteinase-17, were upregulated (both P < 0.03). Since shed syndecan-1 might end up in the circulation, in a stable cross-sectional human renal transplant population (n = 510), we measured plasma syndecan-1. By multivariate regression analysis, we showed robust independent associations of plasma syndecan-1 with renal (plasma creatinine and plasma urea) and endothelial function parameters (plasma VEGF-A, all P < 0.01). By various approaches, we were not able to localize syndecan-1 in vessel wall or endothelial cells, which makes shedding of syndecan-1 from the endothelial glycocalyx unlikely. Our data suggest that early damage in transplanted kidneys induces repair mechanisms within the graft, namely, tubular syndecan-1 expression for tubular regeneration and VEGF production for endothelial repair. Elevated plasma syndecan-1 levels in renal transplantation patients might be interpreted as repair/survival factor related to loss of tubular and endothelial function in transplanted kidneys., (Copyright © 2015 the American Physiological Society.)

Background: Renal aging is characterized by functional and structural changes like decreased glomerular filtration rate, and glomerular, tubular and interstitial damage. To gain insight in pathways involved in renal aging, we studied aged mouse strains and used genetic analysis to identify genes associated with aging phenotypes., Methods: Upon morphological screening in kidneys from 20-month-old mice from 26 inbred strains we noted intracapillary PAS-positive deposits. The severity of these deposits was quantified by scoring of a total of 50 glomeruli per section (grade 0-4). Electron microscopy and immunohistochemical staining for apoE, apoB, apoA-IV and perilipin-2 was performed to further characterize the lesions. To identify loci associated with these PAS-positive intracapillary glomerular deposits, we performed haplotype association mapping., Results: Six out of 26 mouse strains showed glomerular PAS-positive deposits. The severity of these deposits varied: NOD(0.97), NZW(0.41), NON(0.30), B10(0.21), C3 H(0.9) and C57BR(0.7). The intracapillary deposits were strongly positive for apoE and weakly positive for apoB and apoA-IV. Haplotype association mapping showed a strong association with a 30-Kb haplotype block on Chr 1 within the Esrrg gene. We investigated 1 Mb on each site of this region, which includes the genes Spata17, Gpatch2, Esrrg, Ush2a and Kctd3., Conclusions: By analyzing 26 aged mouse strains we found that some strains developed an intracapillary PAS and apoE-positive lesion and identified a small haplotype block on Chr 1 within the Esrrg gene to be associated with these lipoprotein deposits. The region spanning this haplotype block contains the genes Spata17, Gpatch2, Esrrg, Ush2a and Kctd3, which are all highly expressed in the kidney. Esrrg might be involved in the evolvement of these glomerular deposits by influencing lipid metabolism and possibly immune reponses.

Background: Diabetes is associated with a high incidence of macrovascular disease (MVD), including peripheral and coronary artery disease. Circulating soluble-Klotho (sKlotho) is produced in the kidney and is a putative anti-aging and vasculoprotective hormone. Reduced Klotho levels may therefore increase cardiovascular risk in diabetes. We investigated if sKlotho levels are decreased in type 2 diabetes and associate with MVD in the absence of diabetic nephropathy, and whether hyperglycemia affects renal Klotho production in vitro and in vivo., Methods: sKlotho levels were determined with ELISA in diabetic and non-diabetic patients with and without MVD, and healthy control subjects. Human renal tubular epithelial cells (TECs) were isolated and exposed to high glucose levels (15 and 30 mM) in vitro and Klotho levels were measured with qPCR and quantitative immunofluorescence. Klotho mRNA expression was quantified in kidneys obtained from long term (3 and 8 months) diabetic Ins2Akita mice and normoglycemic control mice., Results: No significant differences in sKlotho levels were observed between diabetic patients with and without MVD (527 (433-704) pg/mL, n = 35), non-diabetic MVD patients (517 (349-571) pg/mL, n = 27), and healthy control subjects (435 (346-663) pg/mL, n = 15). High glucose (15 and 30 mM) did not alter Klotho expression in TECs. Long-term hyperglycemia in diabetic Ins2Akita mice (characterized by increased HbA1c levels [12.9 ± 0.3% (3 months) and 11.3 ± 2.0% (8 months)], p < 0.05 vs. non-diabetic mice) did not affect renal Klotho mRNA expression., Conclusions: These data indicate that sKlotho levels are not affected in type 2 diabetes patients with and without MVD. Furthermore, hyperglycemia per se does not affect renal Klotho production. As type 2 diabetes does not alter sKlotho levels, sKlotho does not seem to play a major role in the pathogenesis of MVD in type 2 diabetes.

Background: Ischemia-reperfusion injury (IRI) is a major cause of cardiac damage following various pathological processes. Gaseous hydrogen sulfide (H2S) is protective during IRI by inducing a hypometabolic state in mice which is associated with anti-apoptotic, anti-inflammatory and antioxidant properties. We investigated whether gaseous H2S administration is protective in cardiac IRI and whether non-hypometabolic concentrations of H2S have similar protective properties., Methods: Male C57BL/6 mice received a 0, 10, or 100 ppm H2S-N2 mixture starting 30 minutes prior to ischemia until 5 minutes pre-reperfusion. IRI was inflicted by temporary ligation of the left coronary artery for 30 minutes. High-resolution respirometry equipment was used to assess CO2-production and blood pressure was measured using internal transmitters. The effects of H2S were assessed by histological and molecular analysis., Results: Treatment with 100 ppm H2S decreased CO2-production by 72%, blood pressure by 14% and heart rate by 25%, while treatment with 10 ppm H2S had no effects. At day 1 of reperfusion 10 ppm H2S showed no effect on necrosis, while treatment with 100 ppm H2S reduced necrosis by 62% (p<0.05). Seven days post-reperfusion, both 10 ppm (p<0.01) and 100 ppm (p<0.05) H2S showed a reduction in fibrosis compared to IRI animals. Both 10 ppm and 100 ppm H2S reduced granulocyte-influx by 43% (p<0.05) and 60% (p<0.001), respectively. At 7 days post-reperfusion both 10 and 100 ppm H2S reduced expression of fibronectin by 63% (p<0.05) and 67% (p<0.01) and ANP by 84% and 63% (p<0.05), respectively., Conclusions: Gaseous administration of H2S is protective when administered during a cardiac ischemic insult. Although hypometabolism is restricted to small animals, we now showed that low non-hypometabolic concentrations of H2S also have protective properties in IRI. Since IRI is a frequent cause of myocardial damage during percutaneous coronary intervention and cardiac transplantation, H2S treatment might lead to novel therapeutical modalities.

Genome-wide association studies reported SLC22A2 variants to be associated with serum creatinine. As SLC22A2 encodes the organic cation transporter 2 (OCT2), the association might be due to an effect on tubular creatinine handling. To test this hypothesis we studied the association of SLC22A2 polymorphisms with phenotypes of net tubular creatinine secretion: fractional creatinine excretion (FEcreat) and bias of estimated glomerular filtration rate (eGFR). We also studied the association with end-stage renal disease (ESRD) and graft failure (GF) in renal transplant recipients. SLC22A2 single nucleotide polymorphisms (SNPs), rs3127573 and rs316009, were genotyped in 1,142 ESRD patients receiving renal transplantation and 1,186 kidney donors as controls. GFR was measured with (125)I-iothalamate clearance. Creatinine clearance was also assessed. FEcreat was calculated from the simultaneous clearances of creatinine and (125)I-iothalamate. Donor rs316009 was associated with FEcreat (beta -0.053, P = 0.024) and with estimated [modification of diet in renal disease (MDRD) and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI)] but not measured GFR. In line with this, donor rs316009 was associated with bias of the MDRD and CKD-EPI but not the Cockroft-Gault equation. Both SNPs were associated with ESRD: odds ratios [95% CI] 1.39 [1.16-1.67], P = 0.00065, and 1.23 [1.02-1.48], P = 0.042, for rs3127573 and rs316009, respectively. Neither SNP was associated with GF. Thus, SLC22A2 is associated with phenotypes of net tubular creatinine secretion and ESRD.

Introduction: Lupus nephritis (LN) is a severe and frequent manifestation of systemic lupus erythematosus (SLE). Early detection of initial renal manifestations and relapses during follow-up is pivotal to prevent loss of renal function. Apart from renal biopsies, current urinary and serological diagnostic tests fail to accurately demonstrate the presence of active LN. Previously, we demonstrated that effector memory T-cells (CD45RO+CCR7-;TEM) migrate into the urine during active LN. The objective of this study was to assess the diagnostic value of urinary T-cells in comparison with traditional markers of active LN., Methods: T-cells in the urine during active LN and remission were investigated. Twenty-two, in most cases biopsy-proven, active LN patients and 24 SLE patients without active LN were enrolled and serial measurements were performed in 16 patients., Results: Analysis of the urinary sediment in active renal disease showed an increased number of CD8+ T-cells and absence of these cells during remission. Enumerating T-cell counts in LN patients with a history of renal involvement was a superior marker of active LN in comparison to traditional markers, such as proteinuria and s-creatinine., Conclusions: In conclusion, urinary T-cells, in particular CD8+ T cells, are a promising marker to assess renal activity in LN patients, in particular in those with prior renal involvement.

Background: In recent genetic association studies, common variants including rs12917707 in the UMOD locus have shown strong evidence of association with eGFR, prevalent and incident chronic kidney disease and uromodulin urinary concentration in general population cohorts. The association of rs12917707 with end-stage renal disease (ESRD) in a recent case-control study was only nominally significant., Methods: To investigate whether rs12917707 associates with ESRD, graft failure (GF) and urinary uromodulin levels in an independent cohort, we genotyped 1142 ESRD patients receiving a renal transplantation and 1184 kidney donors as controls. After transplantation, 1066 renal transplant recipients were followed up for GF. Urinary uromodulin concentration was measured at median [IQR] 4.2 [2.2-6.1] yrs after kidney transplantation., Results: The rs12917707 minor allele showed association with lower risk of ESRD (OR 0.89 [0.76-1.03], p = 0.04) consistent in effect size and direction with the previous report (Böger et al, PLoS Genet 2011). Meta-analysis of these findings showed significant association of rs12917707 with ESRD (OR 0.91 [0.85-98], p = 0.008). In contrast, rs12917707 was not associated with incidence of GF. Urinary uromodulin concentration was lower in recipients-carriers of the donor rs12917707 minor allele as compared to non-carriers, again consistent with previous observations in general population cohorts., Conclusions: Our study thus corroborates earlier evidence and independently confirms the association between UMOD and ESRD.

Introduction: Lupus nephritis (LN) is a severe and frequent manifestation of systemic lupus erythematosus (SLE). Its pathogenesis has not been fully elucidated but immune complexes are considered to contribute to the inflammatory pathology in LN. High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different types of cells during activation and/or cell death and may act as a pro-inflammatory mediator, alone or as part of DNA-containing immune complexes in SLE. Urinary excretion of HMGB1 might reflect renal inflammatory injury. To assess whether urinary HMGB1 reflects renal inflammation we determined serum levels of HMGB1 simultaneously with its urinary levels in SLE patients with and without LN in comparison to healthy controls (HC). We also analyzed urinary HMGB1 levels in relation with clinical and serological disease activity., Methods: The study population consisted of 69 SLE patients and 17 HC. Twenty-one patients had biopsy proven active LN, 15 patients had a history of LN without current activity, and 33 patients had non-renal SLE. Serum and urine levels of HMGB1 were both measured by western blotting. Clinical and serological parameters were assessed according to routine procedures. In 17 patients with active LN a parallel analysis was performed on the expression of HMGB1 in renal biopsies., Results: Serum and urinary levels of HMGB1 were significantly increased in patients with active LN compared to patients without active LN and HC. Similarly, renal tissue of active LN patients showed strong expression of HMGB1 at cytoplasmic and extracellular sites suggesting active release of HMGB1. Serum and urinary levels in patients without active LN were also significantly higher compared to HC. Urinary HMGB1 levels correlated with SLEDAI, and showed a negative correlation with complement C3 and C4., Conclusion: Levels of HMGB1 in urine of SLE patients, in particular in those with active LN, are increased and correlate with SLEDAI scores. Renal tissue of LN patients shows increased release of nuclear HMGB1 compared to control renal tissue. HMGB1, although at lower levels, is, however, also present in the urine of patients without active LN. These data suggest that urinary HMGB1 might reflect both local renal inflammation as well as systemic inflammation.

Hydrogen sulfide (H2 S) can induce a reversible hypometabolic state, which could protect against hypoxia. In this study we investigated whether H2 S could protect livers from ischemia/reperfusion injury (IRI). Male C57BL/6 mice were subjected to partial hepatic IRI for 60 min. Animals received 0 (IRI) or 100 ppm H2 S (IRI + H2 S) from 30 min prior to ischemia until 5 min before reperfusion. Core body temperature was maintained at 37° C. Animals were sacrificed after 1, 6 or 24 h. Hepatic ischemia caused extensive hepatic necrosis in the IRI animals which coincided with an increase in ALT and AST serum levels. Animals treated with H2 S showed attenuated serum ALT and AST levels and reduced necrotic lesions after 24 h. IRI animals had increased Bcl-2 mRNA expression and increased active Caspase 3 protein, which were both significantly lower in H2 S treated animals. Increased TNFα and IL-6 mRNA in the IRI livers was significantly attenuated by H2 S treatment, as was hepatic influx of Ly-6G positive granulocytes. Hepatic superoxide production after ischemia was attenuated by H2 S treatment. In hepatic ischemia/reperfusion injury, gaseous H2 S treatment is highly protective, substantially reducing necrosis, apoptosis and inflammation. Gaseous H2 S is therefore a very promising treatment for reducing IRI during hepatic transplantation., (© 2012 The Authors. Transplant International © 2012 European Society for Organ Transplantation.)

Background: Interstitial fibrosis and tubular atrophy (IF/TA) is an important cause of renal function loss and ischaemia-reperfusion (I/R) injury is considered to play an important role in its pathophysiology. The aim of the present study was to investigate the role of a disintegrin and metalloproteinase 17 (ADAM17) in human renal allograft disease and in experimental I/R injury of the kidney., Methods: We studied the expression of ADAM17 messenger RNA (mRNA) in IF/TA and control kidneys by reverse transcription-polymerase chain reaction and in situ hybridization. Moreover, we assessed ADAM17-mediated heparin-binding epidermal growth factor (HB-EGF) shedding in immortalized human cells. Finally, we studied the effect of pharmacological ADAM17 inhibition in a model of renal I/R injury in rats., Results: ADAM17 mRNA was up-regulated in IF/TA when compared to control kidneys. In normal kidneys, ADAM17 mRNA was weakly expressed in proximal tubules, peritubular capillaries, glomerular endothelium and parietal epithelium. In IF/TA, tubular, capillary and glomerular ADAM17 expression was strongly enhanced with de novo expression in the mesangium. In interstitial fibrotic lesions, we observed co-localization of ADAM17 with HB-EGF protein. In vitro, inhibition of ADAM17 with TNF484 resulted in a dose-dependent reduction of HB-EGF shedding in phorbol 12-myrisate 13-acetate-stimulated cells and non-stimulated cells. In vivo, ADAM17 inhibition significantly reduced the number of glomerular and interstitial macrophages at Day 4 of reperfusion., Conclusions: In conclusion, HB-EGF co-expresses with ADAM17 in renal interstitial fibrosis, suggesting a potential interaction in IF/TA. Targeting ADAM17 to reduce epidermal growth factor receptor phosphorylation could be a promising way of intervention in human renal disease.

Syndecan-1, a heparan sulfate proteoglycan, has an important role in wound healing by binding several growth factors and cytokines. As these processes are also crucial in damage and repair after renal transplantation, we examined syndecan-1 expression in human control kidney tissue, renal allograft protocol biopsies, renal allograft biopsies taken at indication, and non-transplant interstitial fibrosis. Syndecan-1 expression was increased in tubular epithelial cells in renal allograft biopsies compared with control. Increased epithelial syndecan-1 in allografts correlated with low proteinuria and serum creatinine, less interstitial inflammation, less tubular atrophy, and prolonged allograft survival. Knockdown of syndecan-1 in human tubular epithelial cells in vitro reduced cell proliferation. Selective binding of growth factors suggests that syndecan-1 may promote epithelial restoration. Bilateral renal ischemia/reperfusion in syndecan-1-deficient mice resulted in increased initial renal failure and tubular injury compared with wild-type mice. Macrophage and myofibroblast numbers, tubular damage, and plasma urea levels were increased, and tubular proliferation reduced in the kidneys of syndecan-1 deficient compared with wild-type mice 14 days following injury. Hence syndecan-1 promotes tubular survival and repair in murine ischemia/reperfusion injury and correlates with functional improvement in human renal allograft transplantation.

Chronic kidney disease (CKD) is a complex disorder. As genome-wide association studies identified cubilin gene CUBN as a locus for albuminuria, and urinary protein loss is a risk factor for progressive CKD, we tested the hypothesis that common genetic variants in CUBN are associated with end-stage renal disease (ESRD) and proteinuria. First, a total of 1142 patients with ESRD, admitted for renal transplantation, and 1186 donors were genotyped for SNPs rs7918972 and rs1801239 (case-control study). The rs7918972 minor allele frequency (MAF) was higher in ESRD patients comparing to kidney donors, implicating an increased risk for ESRD (OR 1.39, p = 0.0004) in native kidneys. Second, after transplantation recipients were followed for 5.8 [3.8-9.2] years (longitudinal study) documenting ESRD in transplanted kidneys--graft failure (GF). During post-transplant follow-up 92 (9.6%) cases of death-censored GF occurred. Donor rs7918972 MAF, representing genotype of the transplanted kidney, was 16.3% in GF vs 10.7% in cases with functioning graft. Consistently, a multivariate Cox regression analysis showed that donor rs7918972 is a predictor of GF, although statistical significance was not reached (HR 1.53, p = 0.055). There was no association of recipient rs7918972 with GF. Rs1801239 was not associated with ESRD or GF. In line with an association with the outcome, donor rs7918972 was associated with elevated proteinuria levels cross-sectionally at 1 year after transplantation. Thus, we identified CUBN rs7918972 as a novel risk variant for renal function loss in two independent settings: ESRD in native kidneys and GF in transplanted kidneys.

Background: Laser microbeam microdissection has greatly facilitated the procurement of specific cell populations from tissue sections. However, the fact that a coverslip is not used means that the morphology of the tissue sections is often poor., Aims: To develop a mounting method that greatly improves the morphological quality of tissue sections for laser microbeam microdissection purposes so that the identification of target cells can be facilitated., Methods: Fresh frozen tissue and formalin fixed, paraffin wax embedded tissue specimens were used to test the morphological quality of mounted and unmounted tissue. The mounting solution consisted of an adhesive gum and blue ink diluted in water. Interference of the mounting solution with DNA quality was analysed by the polymerase chain reaction using 10-2000 cells isolated by microdissection from mounted and unmounted tissue., Results: The mounting solution greatly improved the morphology of tissue sections for laser microdissection purposes and had no detrimental effects on the isolation and efficiency of amplification of DNA. One disadvantage was that the mounting solution reduced the cutting efficiency of the ultraviolet laser. To minimise this effect, the mounting solution should be diluted as much as possible. Furthermore, the addition of blue ink to the mounting medium restores the cutting efficiency of the laser., Conclusions: The mounting solution is easy to prepare and apply and can be combined with various staining methods without compromising the quality of the DNA extracted.

Aim: To report on eight patients with severe idiopathic intermediate uveitis (IU) and granuloma annulare (GA), a self limiting cutaneous condition of unknown aetiology., Methods: Retrospective case series. Clinical ophthalmic and dermatological data were studied and fluorescein angiography and skin biopsies were reviewed., Results: All patients with idiopathic IU had similar ocular features (eight with vitritis, seven with retinal vasculitis) and developed complications such as cystoid macular oedema (n=5), cataract (n=4), and glaucoma (n=3). Systemic diseases were not found, but a localised type of GA was observed in all., Conclusion: Seven out of eight patients with IU and GA developed severe retinal vasculitis. Further studies are needed for a better understanding of this association, a common pathogenesis, and its eventual clinical consequences.

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-zeta (PKC-zeta) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-zeta-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-zeta and Raf-1 is mediated strongly by the 14-3-3beta and -theta; isotypes, but not by 14-3-3zeta. Far-Western blotting revealed that 14-3-3 binds PKC-zeta directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3beta and Raf-1. Treatment of PKC-zeta with lambda protein phosphatase also reduced its binding to 14-3-3beta in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3beta facilitated PKC-zeta binding. Together, the results suggest that 14-3-3 binds both PKC-zeta (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-zeta mutant than with wild-type PKC-zeta, supporting the idea that kinase activity leads to complex dissociation. 14-3-3beta and -&theta; were substrates for PKC-zeta, whereas 14-3-3zeta was not. Phosphorylation of 14-3-3beta by PKC-zeta negatively regulated their physical association. 14-3-3beta with its putative PKC-zeta phosphorylation sites mutated enhanced co-precipitation between PKC-zeta and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-zeta weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-zeta to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-zeta.

Background: Phospholipase D (PLD) hydrolyzes phospholipids to generate phosphatidic acid (PA) and a free headgroup. PLDs occur as both intracellular and secreted forms; the latter can act as potent virulence factors. Exogenous PLD has growth-factor-like properties, in that it induces proto-oncogene transcription, mitogenesis and cytoskeletal changes in target cells. The underlying mechanism is unknown, although it is generally assumed that PLD action is mediated by PA serving as a putative second messenger., Results: In quiescent fibroblasts, exogenous PLD (from Streptomyces chromofuscus) stimulated accumulation of the GTP-bound form of Ras, activation of mitogen-activated protein (MAP) kinase and DNA synthesis, through the pertussis-toxin-sensitive inhibitory G protein Gi. Furthermore, PLD mimicked bioactive lysophospholipids (but not PA) in inducing Ca2+ mobilization, membrane depolarization and Rho-mediated neurite retraction. PLD action was mediated by Iysophosphatidic acid (LPA) derived from Iysophosphatidylcholine acting on cognate G-protein-coupled LPA receptor(s). There was no evidence for the involvement of PA in mediating the effects of exogenous PLD., Conclusions: Our results provide a molecular explanation for the multiple cellular responses to exogenous PLDs. These PLDs generate bioactive LPA from pre-existing Iysophosphatidylcholine in the outer membrane leaflet, resulting in activation of G-protein-coupled LPA receptors and consequent activation of Ras, Rho and Ca2+ signaling pathways. Unscheduled activation of LPA receptors may underlie, at least in part, the known pathogenic effects of exogenous PLDs.

The role of phosphatidylcholine (PC) hydrolysis in activation of the mitogen-activated protein kinase (MAPK) pathway by platelet-derived growth factor (PDGF) was studied in Rat-1 fibroblasts. PDGF induced the transient formation of phosphatidic acid, choline, diacylglycerol (DG), and phosphocholine, the respective products of phospholipase D (PLD) and phospholipase C (PC-PLC) activity, with peak levels at 5-10 min. PLD-catalyzed transphosphatidylation (with n-butyl alcohol) diminished DG formation at 5 min but not at later stages of PDGF stimulation. Phorbol ester-induced down-regulation of protein kinase C (PKC) completely blocked PLD activation but not the formation of DG and phosphocholine at 10 min of PDGF stimulation. Collectively, these data indicate that PDGF activates both PLD and PC-PLC. In contrast, epidermal growth factor did not activate PC-PLC in these cells, and it activated PLD only weakly. DG formation by itself, through Bacillus cereus PC-PLC treatment of cells, was sufficient to mimic PDGF in activation of MAPK independent of phorbol ester-sensitive PKC. Since PKC down-regulation blocked PDGF-induced PLD but not MAPK activation, we conclude that PLD is not involved in MAPK signaling. In contrast, MAPK activation by exogenous (bacterial) PLD was not affected by PKC down-regulation, indicating that signals evoked by exogenous PLD differ from endogenous PLD. D609 (2-10 microg/ml), an inhibitor of PC-PLC, blocked PDGF- but not epidermal growth factor-induced MAPK activation. However, D609 should be used with caution since it also affects PLD activity. The results suggest that PC-PLC rather than PLD plays a critical role in the PDGF-activated MAPK pathway.

Hepatitis B virus (HBV) infection is the world's most important chronic virus infection. No safe and effective treatment is available at present, and clinical exploration of promising antiviral agents, such as nucleoside analogues is hampered because of significant side-effects due to their aspecific body distribution. We are exploring the possibility of the selective delivery of antiviral active drugs to liver parenchymal cells, the main site of infection and replication of HBV. Chylomicrons, which transport dietary lipids into the liver via apolipoprotein E-specific receptors, could serve as drug carriers. However, their endogenous nature hampers their application as pharmaceutical drug carriers. We report here that incorporation of a derivative of the nucleoside analogue iododeoxyuridine into recombinant chylomicrons leads to selective targeting to liver parenchymal cells. Potentially effective intracellular drug concentrations of 700 nM can be achieved, and we therefore anticipate that these drug carrier complexes represent a conceptual advance in the development of an effective and safe therapy for hepatitis B.

Chylomicrons labelled with [3H]cholesterol/[3H]cholesterol esters in a ratio of 25.5: 74.5, were rapidly removed from rat serum in vivo, and taken up predominantly by the parenchymal liver cells (88.2%) of the hepatic uptake at 15 min after injection). Lactoferrin reduced the liver uptake of chylomicron remnants by 72%, at 20 min after injection. It appeared that the free cholesterol which is present in the chylomicrons is not readily exchanged within the used time period with other cholesterol pools in the animal. Between 10-60 min after injection of 3H-labelled chylomicrons, cholesterol esters are hydrolysed in the liver. Appearance of radioactivity in bile was rapid and at 3, 24 and 72 h after injection, 13.4%, 44.0% and 70.0%, respectively, of the injected dose appeared in bile, mainly as bile acids (> 90%). Lactoferrin reduced the biliary secretion of radioactivity, especially during the first hour after injection. The total amount of radioactivity recovered was 58.0% of the injected dose at 72 h after injection. After injection of beta-migrating very low-density lipoprotein labelled with [3H]cholesterol/[3H]cholesterol esters in a ratio of 23.5:76.5, the maximum amount of radioactivity secreted in bile was much lower than with chylomicrons (2.6% cf. 5.2% at 1 h after injection), although the kinetics of the initial liver association and cholesterol ester hydrolysis were even more rapid. Biliary accumulation of radioactivity was also lower with 50.5% of the injected dose recovered at 72 h after injection. It can be concluded from these studies that the processing of chylomicron remnant cholesterol components in the liver and the subsequent secretion in the bile mainly as bile acids is very efficient. The efficient liver uptake of chylomicron remnants by the liver remnant receptor is thereby essential to achieve this high percentage of removal, thus protecting against extrahepatic cholesterol (ester) deposition.

Alpha 2-Macroglobulin (alpha 2M) requires activation by small nucleophiles (e.g. methylamine; giving alpha 2M-Me) or proteolytic enzymes (e.g. trypsin; giving alpha 2M-Tr) in order to be rapidly removed from the circulation by the liver. Separation of rat liver cells into parenchymal, endothelial and Kupffer cells at 10 min after injection indicates that liver uptake of alpha 2M-Me is shared between parenchymal and endothelial cells, with relative contributions of 51.3% and 48.3% respectively of total liver-associated radioactivity. In contrast, alpha 2M-Tr is almost exclusively taken up by the parenchymal cells (90.1% of liver-associated radioactivity). A preinjection of 5 mg of poly(inosinic acid) decreased liver uptake of alpha 2M-Me to 39.9% of the control value, while it had no effect on liver uptake of alpha 2M-Tr. It appears that poly(inosinic acid) specifically reduces the uptake of alpha 2M-Me in vivo by endothelial cells, leaving uptake by parenchymal cells unaffected. In vitro studies with isolated liver cells indicate that the association of alpha 2M-Me with endothelial cells is 21-fold higher per mg of cell protein than with parenchymal cells. The capacity of endothelial cells to degrade alpha 2M-Me appears to be 46 times higher than that of parenchymal cells. Competition studies show that poly(inosinic acid) or acetylated low-density lipoprotein effectively competes with the association of alpha 2M-Me with endothelial and Kupffer cells, but association with parenchymal cells is unaffected. It is suggested that activation of alpha 2M by methylamine induces a charge distribution on the protein which triggers specific uptake by the scavenger receptor on endothelial cells. It is concluded that the uptake of alpha 2M-Me by the scavenger receptor might function as an additional system for the uptake of activated alpha 2M.

The properties of the recognition sites for alpha 2-macroglobulin (alpha 2-macroglobulin receptor; low density lipoprotein receptor-related protein) and beta-migrating very low density lipoprotein (beta-VLDL) (remnant receptor) on rat parenchymal cells were directly compared to analyze whether both substrates are recognized and internalized by the same receptor system. In cholesterol-fed rats, the large circulating pool of beta-VLDL is unable to diminish the liver uptake of 125I-labeled alpha 2-macroglobulin, while liver uptake of 125I-labeled beta-VLDL in these rats is reduced by 87.3% at 10 min after injection. In vitro competition studies with isolated parenchymal liver cells demonstrate that the binding of 125I-labeled alpha 2-macroglobulin to rat parenchymal cells is not effectively competed for by beta-VLDL, whether this lipoprotein is additionally enriched in apolipoprotein E or not. Binding of alpha 2-macroglobulin to parenchymal cells requires the presence of calcium, while binding of beta-VLDL does not. Incubation of parenchymal cells for 1 h with proteinase K reduced the subsequent binding of alpha 2-macroglobulin by 90.1%, while the binding of beta-VLDL was reduced by only 20.2%. In the presence of monensin, the association of alpha 2-macroglobulin to parenchymal cells at 2 h of incubation was reduced by 64.7%, while the association of beta-VLDL was not affected. Preincubation of parenchymal cells with monensin for 60 min at 37 degrees C reduced the subsequent binding of alpha 2-macroglobulin by 54.5%, while binding of beta-VLDL was only reduced by 14.6%. The results indicate that the recognition sites for alpha 2-macroglobulin and beta-VLDL on rat parenchymal cells do exert different properties and are therefore likely to reside on different molecules.

Recently it was found that lactoferrin, an iron-binding glycoprotein with a molecular weight of 76,500, inhibits the remnant receptor-mediated uptake of apolipoprotein E (apoE)-bearing lipoproteins by the liver. In the present study we characterized the hepatic recognition of lactoferrin. Intravenously injected 125I-lactoferrin was cleared rapidly from the circulation by the liver (92.8 +/- 9.5% of the dose at 5 min after injection). Parenchymal cells contained 97.1 +/- 1.5% of the hepatic radioactivity. Internalization, monitored by measuring the release of liver-associated radioactivity by the polysaccharide fucoidin, occurred slowly. Only about 40% of the liver-associated lactoferrin was internalized at 10 min after injection, and it took 180 min to internalize 90%. Subcellular fractionation indicated that internalized lactoferrin is transported to the lysosomes. Binding of lactoferrin to isolated parenchymal liver cells was saturable with a dissociation constant of 10 microM (20 x 10(6) binding sites/cell). The role of arginine residues on lactoferrin was studied by modifying these residues with 1,2-cyclohexanedione. The modification resulted in a strongly reduced liver association (15.9 +/- 1.6% of the dose at 5 min after injection). Furthermore, unlabeled 1,2-cyclohexanedione-modified lactoferrin did not inhibit the binding of 125I-lactoferrin to isolated parenchymal cells. Arginine residues on lactoferrin thus appear to be essential for its specific recognition by parenchymal liver cells. In particular the clustered N-terminal arginine residues, which resemble the arginine-rich receptor binding sequence in apoE, may be responsible for both the interaction of lactoferrin with its recognition site and the inhibition of the hepatic uptake of apoE-bearing lipoproteins.

Upon injection of chylomicrons into rats, chylomicron remnants are predominantly taken up by parenchymal cells, with a limited contribution (8.6% of the injected dose) by Kupffer cells. In vitro storage of partially processed chylomicron remnants for only 24 h leads, after in vivo injection, to an avid recognition by Kupffer cells (uptake up to 80% of the total liver-associated radioactivity). Lactoferrin greatly reduces the liver uptake of chylomicron remnants, which appears to be the consequence of a specific inhibition of the uptake by parenchymal cells. Kupffer-cell uptake is not influenced by lactoferrin. In vitro studies with isolated parenchymal and Kupffer cells show that both contain a specific recognition site for chylomicron remnants. The Kupffer-cell recognition site differs in several ways from the recognition site on parenchymal cells as follows. (a) The maximum level of binding is 3.7-fold higher/mg cell protein than with parenchymal cells. (b) Binding of chylomicron remnants is partially dependent on the presence of calcium, while binding to parenchymal cells is not. (c) beta-Migrating very-low-density lipoprotein is a less effective competitor for chylomicron-remnant binding to Kupffer cells compared to parenchymal cells. (d) Lactoferrin leaves Kupffer-cell binding uninfluenced, while it greatly reduces binding of chylomicron remnants to parenchymal cells. The properties of chylomicron-remnant recognition by parenchymal cells are consistent with apolipoprotein E being the determinant for recognition. It can be concluded that the chylomicron-remnant recognition site on Kupffer cells possesses properties which are distinct from the recognition site on parenchymal cells. It might be suggested that partially processed chylomicron remnants are specifically sensitive to a modification, which induces an avid interaction with the Kupffer cells. The recognition site for (modified) chylomicron remnants on Kupffer cells might function as a protection system against the occurrence of these potential atherogenic chylomicron-remnant particles in the blood.

beta-Migrating very-low-density lipoprotein (beta-VLDL) is a cholesteryl-ester-enriched lipoprotein which under normal conditions is rapidly cleared by parenchymal liver cells. In this study the characteristics of the interaction of beta-VLDL with rat parenchymal cells, Hep G2 cells and human parenchymal cells are evaluated. The binding of beta-VLDL to these cells follows saturation kinetics (Bmax. respectively 117, 106 and 103 ng of beta-VLDL apoliprotein/mg of cell protein), with a relatively high affinity (Kd respectively for beta-VLDL of 10.7, 5.1 and 8.4 micrograms/ml). Competition studies of unlabelled beta-VLDL, low-density lipoprotein (LDL) or acetylated LDL with the binding of radiolabelled beta-VLDL indicate that a LDL-receptor-independent, Ca(2+)-independent, specific recognition site for beta-VLDL is present on rat and human parenchymal cells, whereas with Hep G2 cells or mouse macrophages beta-VLDL recognition is performed by the LDL receptor. The binding of beta-VLDL to Hep G2 cells was down-regulated by 89% by prolonged exposure to beta-VLDL, whereas for human parenchymal and rat parenchymal cells down-regulation of 44% and 20% respectively was observed. Studies with antibodies against the LDL receptor support the presence of a LDL-receptor-independent specific beta-VLDL recognition site on rat and human parenchymal cells. It is concluded that a LDL-receptor-independent recognition site for beta-VLDL is present on rat and human parenchymal liver cells. The presence of a LDL-receptor-independent recognition site on human parenchymal cells may mediate in vivo the uptake of beta-VLDL during consumption of a cholesterol-rich diet, when LDL receptors are down-regulated, thus protecting against the extrahepatic accumulation of the atherogenic beta-VLDL constituents.

The uptake in vivo of chylomicrons and beta-migrating very-low-density lipoprotein (beta-VLDL) by rat liver, which is primarily carried out by parenchymal cells, is inhibited, 5 min after injection, to respectively 35 and 8% of the control values after preinjection of lactoferrin. The decrease in the uptake of lipoproteins by the liver caused by lactoferrin is a specific inhibition of uptake by parenchymal cells. Competition studies in vitro demonstrate that chylomicron remnants and beta-VLDL compete for the same recognition site on parenchymal cells. Data obtained in vivo together with the competition studies performed in vitro indicate that chylomicron remnants and beta-VLDL interact specifically with the same remnant receptor. Hepatic uptake of 125I-labelled-alpha 2-macroglobulin in vivo, mediated equally by parenchymal and endothelial cells, is not decreased by preinjection of lactoferrin and no effect on the parenchymal-cell-mediated uptake is found. In vitro, alpha 2-macroglobulin and chylomicron remnants or beta-VLDL show no cross-competition. Culturing of parenchymal cells for 24-48 h leads to a decrease in the cell association of alpha 2-macroglobulin to 26% of the initial value, while the cell association of beta-VLDL with the remnant receptor is not influenced. It is concluded that beta-VLDL and chylomicron remnants are recognized by a specific remnant receptor on parenchymal liver cells, while uptake of alpha 2-macroglobulin by liver is carried out by a specific receptor system (presumably involving the LDL-receptor-related protein) which shows properties that are distinct from those of the remnant receptor.

A synthetic ribosome-binding site (RBS) containing a 7-nucleotide-long Shine-Dalgarno (SD) sequence was placed ahead of the rat interferon (IFN)-alpha 1 coding region. The translational efficiency of this construct was extremely low. Structural probing of transcripts with RNases T1 and U2 combined with computer predictions revealed the presence of a stable hairpin in which the SD region was base-paired to codons 3, 4 and 5 of the IFN mRNA. Each mutation in this stem changing an A-U to an A.C or a G-C a G.U pair increased translational efficiency about fourfold and this effect could be reversed by a compensating stabilizing substitution in the other strand of the stem. We conclude that the strength of an RBS is to a major degree determined by its involvement in secondary structure. We also show that the negative effect of secondary structure on the efficiency of an RBS can be overcome by allowing upstream translation to terminate within the base-paired region. In our clones, termination-dependent restarts occur at a frequency comparable to that taking place in constructs containing destabilized hairpins.