Human pregnane X receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation.

Delayed graft function (DGF) is an important complication in renal transplantation, contributing significantly to decrease in long-term allograft survival. In addition to donor- and recipient-related risk factors such as immunosuppression, altered renal excretion of xenobiotics by membrane transporters may influence DGF. Using DNA samples from recipients and donors, we assessed the impact on DGF of genetic variants in P-glycoprotein (ABCB1), multidrug resistance protein 2 (ABCC2), and the nuclear pregnane X receptor (PXR/NR1I2), which regulates the transcription of enzymes and transporters. In our local cohort of renal transplant recipients (n = 178), DGF occurred in 27.5%. The PXR 8055TT genotype of the donor only (not of the recipient) was significantly associated with an increased risk for DGF. This finding emerged from univariate as well as multivariate logistic regression analysis including 16 nongenetic factors and held true after correction for multiple testing. Our findings provide the first evidence that PXR may be associated with risk of DGF, independent of previously identified risk factors.

Conditioned medium from renal tubular epithelial cells initiates differentiation of human mesenchymal stem cells.

OBJECTIVES: Mesenchymal-epithelial interactions play a pivotal role in tubular morphogenesis and in maintaining the integrity of the kidney. During renal repair, similar mechanisms may regulate cellular reorganization and differentiation. We have hypothesized that soluble factors from proximal tubular epithelial cells (PTC) induce differentiation of adipose-derived adult mesenchymal stem cells (ASC). This hypothesis has been tested using cultured ASC and PTC. MATERIAL AND METHODS: Conditioned medium was prepared from injured PTC and transferred to ASC cultures. ASC proliferation was analysed by a fluorometric and photometric assay. Signal transduction was analysed by phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2). Grade of ASC differentiation was assessed by morphological analysis and cell expression of characteristic markers. RESULTS: Conditioned medium significantly induced proliferation and phosphorylation of ERK1/ERK2 of ASC. After 12 days of incubation, cell morphology changed to an epithelial-like monolayer. Expression of cytokeratin 18 was induced by conditioned medium, while alpha-smooth muscle actin, CD49a and CD90 expression decreased. These alterations strongly indicate onset of the differentiation process to the epithelial lineage. In summary, soluble factors from PTC induce signal transduction and differentiation of ASC. CONCLUSIONS: Our study shows that conditioned medium from renal tubular epithelial cells provides a convenient source of inductive signals to initiate differentiation of ASC towards epithelial lineage. We deduce that these interactions may play an important role during renal repair mechanisms.

IL-18 is expressed in the intercalated cell of human kidney.

We determined the cellular location of interleukin-18 (IL-18) and caspase-1 and the purinergic receptor P2X7, two proteins necessary for its activation and secretion. The mRNA and protein of IL-18 were detectable in normal human kidney by means of polymerase chain reaction (PCR), in situ hybridization, and Western blot. Immunohistochemistry located IL-18 to nephron segments containing calbinbin-D28k or aquaporin-2 that suggest location in the distal convoluted and the connecting tubule and to parts of the collecting duct. IL-18 was not detected in the thick ascending limb of Henle. Confocal microscopy showed that IL-18 was expressed in cells negative for calbindin-D28k and for aquaporin-2 but positive for the vacuolar H(+)-ATPase. This demonstrates that the intercalated cells produce IL-18. These segments were also positive for caspase-1 and P2X7 that are essential for IL-18 secretion. Our results show that IL-18 is constitutively expressed by intercalated cells of the late distal convoluted tubule, the connecting tubule, and the collecting duct of the healthy human kidney. Since IL-18 is an early component of the inflammatory cytokine cascade, its location suggests that renal intercalated cells may contribute to immediate immune response of the kidney.

C-reactive protein induced activation of MAP-K and RANTES in human renal distal tubular epithelial cells in vitro.

AIMS: C-reactive protein (CRP) is a component of the acute-phase reaction to inflammation, severe tissue injury, and infection. Investigations have shown that CRP concentration is highly increased in the urine during acute renal graft dysfunction and, therefore, may affect tubular cell metabolism. Nevertheless, no data about the effects of CRP on human renal tubular epithelial cells are available. METHODS: Human renal distal tubular cells (DTC) were isolated immunomagnetically and cultured. Cells were stimulated with affinity chromatography pure native CRP from human ascites (10 - 0.001 microg/ml). Phosphorylation of MAP-K was assessed by Westernblot analysis. Release of RANTES and interleukin-6 was evaluated with an enzyme immunoassay. Cytotoxic effects of CRP were determined by a commercially available Live/Dead assay and MTT assay. Effects on cell proliferation were analyzed by a fluorimetric assay. RESULTS: Westernblot analysis clearly showed that CRP activates the MAP-K pathway of DTC. CRP upregulated RANTES expression of DTC in a significant and dose-dependent manner. CRP (10 microg/ml) induced a 12.3-fold upregulation, CRP 1 or 0.1 microg/ml induced a 6.3-/2.8-fold RANTES upregulation, respectively. Interleukin-6 synthesis was not influenced. Cytotoxic, proliferative or apoptotic effects were not observed at the concentrations used. CONCLUSIONS: We demonstrated an activating effect of CRP on DTC in vitro. In vivo, this effect of CRP might be part of the immune activation cascade during episodes of renal graft rejection or bacterial infections.

Epstein-barr virus-associated posttransplant lymphoproliferative disorder of donor origin after simultaneous pancreas-kidney transplantation limited to pancreas allograft: A case report.

A 45-year-old man was admitted with fever and elevated pancreas enzymes 6 months after simultaneous pancreas-kidney transplantation (SPKT). Function of the allografts was normal. Bacterial and fungal infections were excluded, while Epstein-Barr virus (EBV)-polymerase chain reaction (PCR) was positive. However, screening for EBV-associated lymphoma was negative. EBV infection did not respond to antiviral therapy. After an 18F-Fluorodeoxyglucose positron emission tomography positive signal and an abnormal computed tomography scan of the pancreas transplant, a biopsy revealed a diffuse large monomorphic B-cell lymphoma, which was confined to the grafted organ. Its origin was assigned to the donor by microsatellite analysis. Reduction of immunosuppression and immunotherapy with rituximab was unsuccessful. After 10 weeks, the patient developed an acute hemolytic uremic syndrome which required explantation of the allografts. Subsequent to the intervention, fever disappeared, EBV DNA became undetectable and lymphoma screening remained negative. In posttransplant lymphoproliferative disorder of donor origin after SPKT, transplantectomy may be a curative therapy.

Expression of osteopontin (Eta-1) in Crohn disease of the terminal ileum.

BACKGROUND: The causes of Crohn disease (CD) are still regarded as unknown, but impaired mucosal immunoregulation with activation of T-helper-1 (Th-1) cytokine responses is probably involved and may contribute to the morphological changes. We investigated a possible role of osteopontin (Opn) in the pathogenesis of CD. This glycoprotein has been suggested to be involved in the generation of Th-1-type immune responses; moreover, it carries anti-inflammatory activities. METHODS: Ileal samples from CD patients--both actively inflamed and inactive areas as well as unaffected intestinal specimens from controls (normal ileum)--were investigated by Western blot analysis, immunohistochemistry and in situ hybridization. RESULTS: In normal gut, Opn was found to be regularly expressed by plasma cells (CD 38) and a subset of lamina propria mononuclear cells (MNC) as well as by intestinal epithelial cells (IEC). In active CD, immunohistochemistry and in situ hybridization analysis revealed a loss of Opn expression by IEC adjacent to ulcerative lesions, whereas especially plasma cells (CD 38) in the vicinity of such lesions were found to express the molecule. In addition, a slight overexpression of Opn protein was found in metaplastic crypts. However, quantitative analysis of total Opn protein in the ileal mucosa of CD patients did not reveal any difference vis-à-vis control tissues. CONCLUSIONS: The constitutive expression of Opn in normal gut indicates that it is involved in intestinal immune homeostasis. Downregulation of Opn expression in IEC might favour the disintegration of the epithelial barrier. The expression of Opn in lamina propria plasma cells could contribute to disease chronification, probably by affecting cell survival.

Glomerular osteopontin expression and macrophage infiltration in glomerulosclerosis of DOCA-salt rats.

Expression of the chemoattractant osteopontin (OPN) may contribute to macrophage infiltration in many types of tubulointerstitial kidney disease, but the role of OPN in chronic glomerulosclerosis is unknown. We hypothesized that glomerular OPN expression and macrophage infiltration occur in deoxycorticosterone acetate (DOCA)-salt glomerulosclerosis in rats. Uninephrectomized rats receiving DOCA pellets and 1% saline were compared with control rats. OPN mRNA was determined by Northern blot, and OPN protein was determined by Western blot. The localization of OPN was studied by in situ hybridization and double immunohistochemistry with glomerular cell markers. Macrophage infiltration was quantified by counting ED-1-positive cells, and semiquantitative glomerulosclerosis scores were obtained. In DOCA-salt rats, OPN mRNA in the kidney was increased 2-fold over control after 9 days and 3 weeks and 20-fold after 6 weeks. Tubulointerstitial OPN staining was apparent after 21 days of DOCA treatment. Glomerular OPN mRNA and protein was detected after 42 days in parietal and visceral epithelial cells, activated myofibroblasts, and occasionally mesangial cells. Progressive glomerular macrophage infiltration occurred during the development of DOCA hypertension, paralleling the degree of glomerulosclerosis. Glomeruli staining positive for osteopontin contained more macrophages (18.4 +/- 3.4 per cross-section) than osteopontin-negative glomeruli (3.6 +/- 0.5; P < 0.05). Glomerular OPN expression occurs in chronic hypertensive glomerulosclerosis and is associated with macrophage infiltration. The data suggest a role for OPN as a chemoattractant in hypertensive glomerulosclerosis.

Osteopontin is expressed by adult human osteoarthritic chondrocytes: protein and mRNA analysis of normal and osteoarthritic cartilage.

Osteopontin, a sulfated phosphoprotein with cell binding and matrix binding properties, is expressed in a variety of tissues. In the embryonic growth plate, osteopontin expression was found in bone-forming cells and in hypertrophic chondrocytes. In this study, the expression of osteopontin was analyzed in normal and osteoarthritic human knee cartilage. Immunohistochemistry, using a monoclonal anti-osteopontin antibody was negative on normal cartilage. These results were confirmed in Western blot experiments, using partially purified extracts of normal knee cartilage. No osteopontin gene expression was observed in chondrocytes of adult healthy cartilage, however, in the subchondral bone plate, expression of osteopontin mRNA was detected in the osteoblasts. In cartilage from patients with osteoarthritis, osteopontin could be detected by immunohistochemistry, Western blot analysis, in situ hybridization, and Northern blot analysis. A qualitative analysis indicated that osteopontin protein deposition and mRNA expression increase with the severity of the osteoarthritic lesions and the disintegration of the cartilaginous matrix. Osteopontin expression in the cartilage was limited to the chondrocytes of the upper deep zone, showing cellular and territorial deposition. The strongest osteopontin detection was found in deep zone chondrocytes and in clusters of proliferating chondrocytes from samples with severe osteoarthritic lesions. These data show the expression of osteopontin in adult human osteoarthritic chondrocytes, suggesting that chondrocyte differentiation and the expression of differentiation markers in osteoarthritic cartilage resembles that of epiphyseal growth plate chondrocytes.

Effects of mycophenolic acid on human renal proximal and distal tubular cells in vitro.

BACKGROUND: Mycophenolic acid has been shown to be effective for the prevention and treatment of renal allograft rejection. Rejection episodes were found to be associated with an infiltration of lymphocytes and macrophages/monocytes into the diseased kidney. Expression of RANTES, HLA-DR and ICAM-1 may be important for the pathogenesis of this leukocyte infiltration. Therefore the aim of this study was to evaluate the effect of the antiproliferative and immunosuppressive agent mycophenolic acid (MPA) on cell growth and cytokine-induced expression of RANTES, HLA-DR and ICAM-1 of highly purified proximal (PTC) and distal tubular cells (DTC) from human kidney. METHODS: Human PTC and DTC were cultured in the presence of different concentrations of MPA (0.25-50 microM) or MPA plus guanosine (100 microM). Total cell number (DNA content) was determined after 4 days of cell culture by a non-radioactive fluorescence assay. Cells were stimulated by a combination of cytokines (IL1beta+gammaIFN+TNFalpha=cytomix) or cytomix plus MPA. Secretion of RANTES protein was evaluated with an enzyme-linked-immunosorbent assay. Cell surface expression of HLA-DR and ICAM-1 was assessed by flow cytometric analysis. RESULTS: MPA inhibited cell growth of PTC and DTC in a dose-dependent manner. This effect was totally abolished by the addition of guanosine. Cytokine-induced RANTES expression was synergistically increased in the presence of MPA, an effect that was partially prevented by the addition of guanosine. Cytokine stimulation resulted in de novo expression of HLA-DR and a marked increase of ICAM-1 expression, which was partially inhibited by dexamethasone. Addition of MPA did not influence this stimulated expression. CONCLUSIONS: We demonstrate that MPA has an effect on cell growth and chemokine release of tubular epithelial cells, and that these effects are dependent on the inhibition of cellular guanosine production. The clinical consequences of this possible pro-inflammatory effect of MPA on RANTES release may be abolished by a concomitant treatment with steroids.

Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells.

UNLABELLED: Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells. BACKGROUND: High-output levels of nitric oxide (NO) are produced by rat mesangial cells (MCs) in response to proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) by the inducible isoform of NO synthase (iNOS). We tested modulatory effects of NO on the expression and activities of matrix metalloproteinases-9 and -2 (MMP-9 and MMP-2), respectively. Temporal and spatial expression of these MMPs and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), seems to be critical in the extensive extracellular matrix (ECM) remodeling that accompanies sclerotic processes of the mesangium. Methods and Results. Using the NO donors S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETA-NONOate, we found strong inhibitory effects of NO mainly on the IL-1beta-induced MMP-9 mRNA levels. NO on its own had only weak effects on the expression of MMP-9 and MMP-2. The addition of the NOS inhibitor NG-monomethyl L-arginine (L-NMMA) dose dependently increased steady-state mRNA levels of cytokine-induced MMP-9, suggesting that endogenously produced NO exerts tonic inhibition of MMP-9 expression. MMP-9 activity in conditioned media from MCs costimulated with IL-1beta and NO donor contained less gelatinolytic activity than media of cells treated with IL-1beta alone. Exogenously added NO did not alter gelatinolytic activity of MMP-9 in cell-free zymographs. The expression levels of TIMP-1 were affected by NO similarly to the expression of MMP-9. CONCLUSION: We conclude that NO modulates cytokine-mediated expression of MMP-9 and TIMP-1 in rat MCs in culture. Our results provide evidence that NO-mediated attenuation of MMP-9 gelatinolytic activity is primarily due to a reduced expression of MMP-9 mRNA, and not the result of direct inhibition of enzymatic activity.

Exogenous nitric oxide inhibits mesangial cell adhesion to extracellular matrix components.

Interactions of mesangial cells (MCs) with components of the extracellular matrix (ECM) profoundly influence the MC phenotype, such as attachment, contraction, migration, survival and proliferation. Here, we investigated the effects of exogenous nitric oxide (NO) on the process of MC adhesion to ECM molecules. Incubation of rat MCs with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) dose- and time-dependently inhibited MC adhesion and spreading on various ECM substrata, being more pronounced on collagen type I than on collagen type IV, laminin or fibronectin. In contrast, SNAP did not inhibit MC adhesion to L-polylysine-coated plates. The inhibitory effects of SNAP were reduced by hemoglobin and enhanced by superoxide dismutase. The anti-adhesive action of SNAP was mimicked not only by other NO donors but also by 8-bromo-cGMP, and significantly reversed by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha]quinoxalin-1-one (ODQ). Moreover, SNAP and 8-bromo-cGMP decreased the adhesion-induced phosphorylation of focal adhesion kinase (pp125FAK). In the presence of SNAP or 8-bromo-cGMP, adherent MCs exhibited disturbed organization of alpha-actin filaments and reduced numbers of focal adhesions, as shown by immunocytochemistry. In additional experiments with adherent MCs, it was found that exposure to SNAP or 8-bromo-cGMP for 12 and 24 hours induced detachment of MCs. The results indicate that exogenous NO interferes with the establishment and maintenance of MC adhesion to ECM components. This inhibitory NO effect is mediated predominantly by cGMP-signaling. Disturbance of MC attachment to ECM molecules could represent an important mechanism by which NO affects MC behavior in vitro and in vivo.

Adhesion molecules in the glomerular mesangium.

Experimental evidence indicates that extensive "cross-talk" exists between glomerular cells, extracellular matrix molecules and soluble mediator substances affecting the proliferative and secretory phenotype of glomerular mesangial cells. Both matrix and cytokines regulate mesangial cell behavior in vitro and in vivo after binding to specific cell surface receptors. It appears as if the concerted action of insoluble and soluble ligands on mesangial cells involves a reciprocal regulation of matrix molecules and cytokines as well as expression and affinity of their respective receptors. Elucidation of the potential biologic and clinical relevance of cell-matrix interactions in the glomerular mesangium represents a challenging goal in current kidney research. This brief review summarizes recent investigations concerning regulation of expression and function of adhesion molecules and matrix receptors in the mesangium. In addition to results from cell culture studies, descriptive findings on expression and regulation of adhesion molecules and their potential role for altered mesangial cell behavior in glomerular disease is considered.

Glomerular basement membrane-derived perlecan inhibits mesangial cell adhesion to fibronectin.

To explore whether the heparan sulfate proteoglycan, periecan, may act as a regulator of glomerular cell behavior, we examined the effects of perlecan on adhesive properties of rat and human mesangial cells in culture. We observed that mesangial cells did not adhere to purified perlecan. Mesangial cell adhesion to fibronectin, but not to laminin, was inhibited when perlecan was present in the substratum, reaching complete inhibition at 20 micrograms/ml perlecan. Similarly, perlecan reduced mesangial cell adhesion to the 120 kDa fragment of fibronectin and to bovine serum albumin-coupled RGD peptides both lacking the fibronectin heparin-binding domains, indicating that the inhibitory effect of perlecan does not require interaction of its heparan sulfate chains with fibronectin. The core protein of perlecan seems to be relevant, since perlecan retained most of its anti-adhesive potency after total deglycosylation. It is unlikely that perlecan causes steric inhibition of the cell binding sites of fibronectin by direct binding, since the 120 kDa fragment of fibronectin which includes the cell binding site, did not bind to intact perlecan. In the presence of submaximal inhibitory concentrations of perlecan, soluble RGD peptides (100 micrograms/ml), by themselves without effect, completely inhibited cell adhesion to fibronectin. Using immunocytochemistry, we examined the effects of perlecan on the organization of fibronectin-specific, RGD-dependent beta 1 integrins in focal contacts. When perlecan was added to the substratum, human mesangial cells adhered to fibronectin only in the presence of collagen I. Under these conditions, perlecan did not significantly alter the organization of alpha 5 beta 1 integrin into focal contacts of mesangial cells which adhered to a substratum composed of collagen I plus fibronectin. These data suggest that perlecan exerts its anti-adhesive effects on mesangial cells via a core protein-dependent mechanism which reduces the avidity of the fibronectin receptor. The resulting avidity level is too low for mediating cell binding but sufficient to induce integrin organization into focal contacts.

Mesangial cell-matrix interactions in glomerular inflammation.

Specific interactions between cells and components of the surrounding extracellular matrix (ECM) or underlying basement membrane have been shown to modulate cell behaviour, in culluro and in vivo. There is evidence that extensive 'cross-talk' occurs between glomerular mesangial cells (MCs), ECM molecules, and soluble mediator substances affecting the proliferative and synthetic-secretory phenotype of MCs. This is likely to be relevant for the behaviour of MCs during embryonic development, disease processes of glomeruli, and tissue repair. The potential biologic and clinical relevance of cell-matrix interactions in the glomerulus are discussed in this brief review of selected aspects of recent investigations concerning the mesangial matrix and its interactions with MCs.

Inhibition of proliferation of human cerebral meningioma cells by suramin: effects on cell growth, cell cycle phases, extracellular growth factors, and PDGF-BB autocrine growth loop.

The growth of human cerebral meningiomas depends on various growth factors, including epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and TGF-beta, platelet-derived growth factor (PDGF)-BB, insulin-like growth factor (IGF)-I and IGF-II, and acidic and basic fibroblast growth factors. The latter three have been shown to form autocrine loops that are thought to be a major component of uncontrolled growth in meningioma tissue. Suramin is known to prevent binding of a variety of growth factors to their receptors in mammalian tissue, thus abolishing para- and/or autocrine-mediated cell growth. The authors therefore tested the effect of suramin on the proliferation of cultured human meningioma cells. Suramin (10(-5) to 10(-4) M) significantly inhibited the growth of meningioma cells in culture. The maximum effect observed was with the higher dose (10(-4) M), which resulted in a 40% to 70% reduction in cellular proliferation. This effect was observed in all 15 tumor samples studied and was confirmed by [3H]thymidine uptake. In studies using DNA flow cytometry, suramin inhibited meningioma cell proliferation in five tumor samples by arresting cells in the S and G2/M phases of the cell cycle. Growth factor (EGF, IGF-I, and PDGF-BB)-induced cell proliferation was completely abolished in five tumor samples when 10(-4) M suramin was applied to meningioma cells. Western blot analysis of three tumor samples showed that the intracellular PDGF-BB content of meningioma cells was significantly reduced after treating the cells with 10(-4) M suramin. Binding of iodinated growth factors (that is, [125I]EGF, [125I]IGF-I, and [125I]PDGF-BB) to their receptor sites was prevented by suramin in a dose-dependent manner in 10 meningioma membrane fractions. Lowering of the intracellular PDGF content and prevention of extracellular growth factor receptor binding demonstrates that suramin disrupts autocrine loops and paracrine growth stimulation in meningioma tissue. These data provide evidence that growth of cerebral meningiomas in culture is strongly inhibited by suramin at a concentration of 10(-4) M. Suramin acts as a scavenger neutralizing exogenous growth factors; thus it can interrupt autocrine loops and paracrine stimulation of human meningioma cell growth. The evidence favors suramin as a therapeutic option for controlling meningioma proliferation in patients with inoperable and recurrent high-grade meningiomas.

[Deficits in school performance in children and adolescents with chronic asthma]. / Schuldefizite chronisch asthmakranker Kinder und Jugendlicher.

At the time of their admission to the Buchenhöhe Jugendorf asthma centre (an institution for medical, professional and social rehabilitation), an investigation was made into the cases of 186 children and youths (average age: 14.0), the majority of whom suffered from severe asthma. The investigation, focused on the patients' ailment symptomatology and its effect on school progress, showed that one third of the children had spent considerable time hospitalized in intensive care (an average of 4.4 x). Amongst other findings were: That one third on the children and youths had not taken part in sports, either at school or in their leisure time, that they had missed a lot of school (33 children had missed over 30%) and that the children and youths were an average of 1.29 years behind the normal school level. The investigation leads to the conclusion that the schooling situation of asthmatic children and youths should be taken into consideration within the chronic asthma treatment plan and that treatment periods that last too long bring about an irreversible school deficiency which could hinder the further development of the youth.