Intrarenal urothelium proliferation: an unexpected early event following ischemic injury.

Identification of renal cell progenitors and recognition of the events contributing to cell regeneration following ischemia-reperfusion injury (IRI) are a major challenge. In a mouse model of unilateral renal IRI, we demonstrated that the first cells to proliferate within injured kidneys were urothelial cells expressing the progenitor cell marker cytokeratin 14. A systematic cutting of the injured kidney revealed that these urothelial cells were located in the deep cortex at the corticomedullary junction in the vicinity of lobar vessels. Contrary to multilayered bladder urothelium, these intrarenal urothelial cells located in the upper part of the medulla constitute a monolayered barrier and express among uroplakins only uroplakin III. However, like bladder progenitors, intrarenal urothelial cells proliferated through a FGF receptor-2 (FGFR2)-mediated process. They strongly expressed FGFR2 and proliferated in vivo after recombinant FGF7 administration to control mice. In addition, IRI led to FGFR phosphorylation together with the selective upregulation of FGF7 and FGF2. Conversely, by day 2 following IRI, renal urothelial cell proliferation was significantly inhibited by FGFR2 antisense oligonucleotide administration into an intrarenal urinary space. Of notice, no significant migration of these early dividing urothelial cells was detected in the cortex within 7 days following IRI. Thus our data show that following IRI, proliferation of urothelial cells is mediated by the FGFR2 pathway and precedes tubular cell proliferation, indicating a particular sensitivity of this structure to changes caused by the ischemic process.

Transforming growth factor-beta inhibits interleukin-10 synthesis by human monocytic cells.

Transforming growth factor-beta (TGF-beta1) enhances interleukin-10 (IL-10) synthesis by mouse monocytes/macrophages, suggesting a potential role of IL-10 in mediating some of the anti-inflammatory properties of TGF-beta1. Since differences exist between the transcriptional regulation of human and mouse IL-10, the studies reported here examined whether TGF-beta1 up-regulated IL-10 production by human monocytes/macrophages as well. Exposure of PMA-differentiated U-937 promonocytic cells to TGF-beta1 resulted in an unexpected, dose-dependent decrease in IL-10 production as assessed by specific ELISA. TGF-beta1 was effective when added at the time of the PMA stimulus or 6 hours after. In addition, TGF-beta1 suppressed induction of IL-10 by three different stimuli other than PMA. TGF-beta1 inhibition of IL-10 protein release was associated with proportional changes in IL-10 mRNA accumulation as assessed by quantitative kinetic ELISA PCR. This would result from a decrease in IL-10 gene transcription as TGF-beta1 did not affect IL-10 mRNA stability, and TGF-beta1 limited the luciferase activity in cells transfected with reporter gene constructs containing 1,308 bp of the 5' non-coding sequence of human IL-10 gene. Blocking tumour necrosis factor-alpha (TNF-alpha) with neutralizing anti-TNF-alpha antibody did not modify the response to TGF-beta1, indicating the involvement of TNF-alpha-independent mechanisms in the overall process. Thus, the present study provides the first evidence that TGF-beta1 prevents IL-10 production by human monocytic cells at a transcriptional level.

Cytokines and hormones with anti-inflammatory effects: new tools for therapeutic intervention.

The inflammation that is involved in the development of glomerulonephritis is tightly regulated by the expression of anti-inflammatory factors. These include circulating hormones, such as glucocorticoids, and mediators that are produced by intrinsic cells and infiltrating leucocytes. The present review focuses on these anti-inflammatory factors, summarizing in particular their activities in existing models of glomerulonephritis. In addition, experimental evidence is presented that anti-inflammatory mediators are able to increase glucocorticoid binding or signalling in target cells. These data help to explain the in-vivo efficacy of anti-inflammatory mediators, and offer a promising new avenue for therapeutic intervention.

Glomerulosclerosis in mice transgenic for human insulin-like growth factor-binding protein-1.

BACKGROUND: The growth hormone (GH)/insulin-like growth factor (IGF) system is thought to participate in the glomerulosclerosis process. Because IGF-binding proteins (IGFBPs) modulate IGF actions and hence GH secretion, this study assessed whether mice transgenic for human IGFBP-1 have altered susceptibility to glomerulosclerosis. METHODS: A line of transgenic mice that express human IGFBP-1 mRNA in the liver under the control of the alpha1-antitrypsin promoter has been obtained, and morphological changes in the kidney tissue were assessed. Glomerulosclerosis was identified using light microscopy, light microscopic morphometry, and electron microscopy. Extracellular matrix components were analyzed by immunohistochemistry. RESULTS: There was a marked increase in mesangial extracellular matrix area in homozygous transgenic mice at three months of age as compared with heterozygous transgenic mice and nontransgenic littermates. These changes were not associated with alterations in glomerular volume or cellularity. The expansion of extracellular matrix area was related to a marked increase in laminin and type IV collagen and to the appearance of type I collagen. CONCLUSIONS: These observations indicate that the enhanced expression of IGFBP-1 may result in the development of glomerulosclerosis without glomerular hypertrophy. The changes are potentially related to a decrease in IGF-I availability and/or to an IGF-I-independent role of IGFBP-1.

Somatostatin increases glucocorticoid binding and signaling in macrophages by blocking the calpain-specific cleavage of Hsp 90.

Somatostatin has direct anti-inflammatory actions and participates in the anti-inflammatory actions of glucocorticoids, but the mechanisms underlying this regulation remain poorly understood. The objective of this study was to evaluate whether somatostatin increases glucocorticoid responsiveness by up-regulating glucocorticoid receptor (GR) expression and signaling. Somatostatin promoted a time- and dose-dependent increase in [(3)H]dexamethasone binding to RAW 264.7 macrophages. Cell exposure to 10 nM somatostatin for 18 h promoted a 2-fold increase in the number of GR sites per cell without significant modification of the affinity. Analysis of GR heterocomplex components demonstrated that somatostatin increased the level of heat shock protein (Hsp) 90, whereas the level of GR remained almost unchanged. The increase in Hsp 90 was associated with a decrease in the cleavage of its carboxyl-terminal domain. Evidence for the involvement of calpain inhibition in this process was obtained by the demonstration that 1) somatostatin induced a dose-dependent decrease in calpain activity and 2) calpain inhibitors, calpain inhibitor I and calpeptin, both abolished the cleavage of Hsp 90 and induced a dose-dependent increase in [(3)H]dexamethasone binding. Increases in glucocorticoid binding after somatostatin treatment were associated with similar increases in the ability of GR to transactivate a minimal promoter containing two glucocorticoid response elements (GRE) and to interfere with the activation of nuclear factor-kappaB (NF-kappaB). Thus, the present findings indicate that somatostatin increases glucocorticoid binding and signaling by limiting the calpain-specific cleavage of GR-associated Hsp 90. This mechanism may represent a novel target for intervention to increase glucocorticoid responsiveness.

Reactive oxygen and nitrogen intermediates increase transforming growth factor-beta1 release from human epithelial alveolar cells through two different mechanisms.

Transforming growth factor (TGF)-beta1 is a growth factor involved in the mechanisms of lung repair and fibrosis that follow inflammatory processes. We sought to examine the link between the generation of reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI) by inflammatory cells and the expression of TGF-beta1 by alveolar epithelial cells. Exposure of the A549 lung epithelial cell line to either an ROI generating system (xanthine and xanthine oxidase) or an RNI donor (S-nitroso-N-acetyl-penicillamine [SNAP]) promoted a time- and dose-dependent increase in TGF-beta1 release, as measured by a specific enzyme-linked immunosorbent assay. At the peak, the levels of TGF-beta1 were twice the control values. The induction of TGF-beta1 release by ROI was blunted by catalase and unaffected by superoxide dismutase, indicating the involvement of hydrogen peroxide. The response was also blunted by 5, 6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), a specific RNA polymerase II inhibitor, and accompanied by a corresponding increase in TGF-beta1 messenger RNA, as measured by quantitative/competitive reverse transcription polymerase chain reaction, suggesting the involvement of transcriptional mechanisms and possibly other downstream mechanisms. In contrast, RNI-induced TGF-beta1 release was unaffected by DRB and blunted by the protein synthesis inhibitor cycloheximide, suggesting the involvement of translational and post-translational mechanisms. This response required cyclic guanosine monophosphate (cGMP)- mediated processes because (1) immunoreactive cGMP accumulated in the culture medium of SNAP-treated cells; (2) SNAP-induced TGF-beta1 release was blunted by KT 5823, an inhibitor of cGMP-dependent protein kinase; and (3) similar increase in TGF-beta1 release was obtained by cell exposure to membrane-permeable dibutyryl-cGMP or to atrial natriuretic factor, a known agonist of particulate guanylate cyclase. These data suggest that in vitro exposure of human alveolar epithelial cells to ROI and RNI enhances TGF-beta1 release through different mechanisms. In vivo, this control may constitute a molecular link between inflammatory and fibrotic processes.

[Inflammatory mechanisms of renal fibrosis: glomerulonephritis]. / Mécanismes inflammatoires de la fibrose rénale: les glomérulonéphrites.

Studies of glomerulonephritis models have shown that inflammatory reaction is responsible for the development of glomerulosclerosis and tubulo-interstitial sclerosis and, hence, for the progression to end stage renal failure. That macrophage accumulation and fibrosis extension are frequently not closely related events suggests that macrophages are not involved in progression process. Glomerular sclerosis is rather associated with the release of mediators from resident cells-mainly growth factors such as platelet-derived growth factor and transforming growth factor-beta--the synthesis and bioactivity of which are enhanced by inflammatory mediators. Tubulo-interstitial sclerosis is induced by inflammatory lesions of the glomerulus that lead to proteinuria. Indeed, reabsorption of proteins in proximal tubule triggers epithelial cells to release proinflammatory and prosclerotic mediators into the interstitium. New therapeutic approaches including gene transfer strategies are directed at suppressing the efficiency of such mediators.

Growth hormone and somatostatin in glomerular injury.

Among other neuropeptides and neurohormones, growth hormone (GH) and somatostatin (SRIF) have been shown to modulate the development of glomerular injury in various renal diseases. In particular, GH is implicated in the induction of glomerular hypertrophy and sclerosis in partial nephrectomy and diabetic nephropathy. While GH effects on glomerular hypertrophy are likely mediated by insulin-like growth factor I (IGF-I), GH effects on glomerular sclerosis are independent of IGF-I. Those effects rather require multiple signaling pathways functioning in series, e.g. angiotensin II binding preceding transforming growth factor beta (TGF-beta) release, or pro-inflammatory factor release preceding repair/scarring processes. In contrast with GH, SRIF administration prevents the development of glomerular lesions in experimental diabetes, partial nephrectomy and immune glomerulonephritis. Inhibitory effects of SRIF on glomerular hypotrophy may be through a decrease in GH secretion and/or IGF-I expression or through a direct blockade of glomerular cell proliferation. The mechanisms underlying the anti-inflammatory effects of SRIF are most likely a deactivation of inflammatory cells related in part to an upregulated response of these cells to glucocorticoids. Additional studies will be required to further define the role of GH and SRIF in the development of glomerular injury and, hence, to identify new targets for a therapeutic approach in glomerular diseases.

Reduction of tumour necrosis factor alpha expression and signalling in peripheral blood mononuclear cells from patients with thalassaemia or sickle cell anaemia upon treatment with desferrioxamine.

Recent evidence indicates that the rate of progression of the HIV-1 disease is significantly reduced in thalassaemia major patients upon treatment with high doses of desferrioxamine (DFX). The authors have previously demonstrated that in vitro exposure of mononuclear cells to DFX decreases the bioavailability of tumour necrosis factor alpha (TNF-alpha) which has a stimulatory effect on HIV-1 replication. In this study, therefore, TNF-alpha bioavailability from mononuclear cells isolated from 10 patients with thalassaemia or sickle cell anaemia given DFX as compared to 10 untreated subjects has been evaluated. Evidence is presented showing that DFX treatment reduces TNF-alpha bioavailability (P<0.05) by inhibiting its steady state (P<0.05) and by enhancing its inactivation through binding to soluble TNF-alpha receptor type II (P<0.05). We also show that DFX treatment limits the in vivo activation of NF-kappaB, a transcription factor involved in both TNF-alpha gene transcription and TNF-alpha signalling (P<0.005). We conclude that TNF-alpha bioavailability and signalling are impaired in patients upon DFX treatment. This mechanism may contribute to delayed progression of the HIV-1 infection in vivo.

[Interactions between glucocorticoids and anti-inflammatory peptides]. / Interactions entre glucocorticoïdes et peptides anti-inflammatoires.

Both pro- and anti-inflammatory mediators regulate the anti-inflammatory actions of glucocorticoids, in part by modifying the binding of glucocorticoids to specific receptors. For instance, somatostatin has been shown to increase glucocorticoid binding and signaling in macrophages. The mechanism of this regulation does not require an increased expression of glucocorticoid receptors but, rather, a stabilization of glucocorticoid receptor-associated heat shock protein 90. This is related to a decrease in calpain activity. Thus calpain inhibition may offer a new and exciting possibility for enhancing the anti-inflammatory efficiency of glucocorticoids.

[Polyuropolydipsic syndromes]. / Syndromes polyuropolydipsiques.

PROGNOSIS: Intracellular dehydration is the major risk in case of a polyuropolydipsic syndrome. Excepting osmotic polyuria, prognosis depends on a possibly progressive functional anomaly of the hypothalamopituitary axis. PATHOPHYSIOLOGY: Polyuropolydipsia occurs when antidiuretic hormone (ADH) secretion is absent (central diabetes insipidis), the kidney does not respond to ADH (nephrogenic diabetes insipidus) or in case of physiological inhibition of ADH secretion (primary polydipsia). EXPLORATION: Dynamic explorations are associated with radioimmunoassay of ADH. They are particularly useful in case of atypical diabetes insipidus and include the water restriction test and a study of the sensitivity to exogenous ADH (dDAVP). The results orient the etiologic diagnosis and allow an evaluation of the fluid intake required as a function of the maximal concentrating capacity of the kidneys. TREATMENT OF CENTRAL DIABETES INSIPIDUS: Treatment is based on ADH analogs (dDAVP). The aim is to obtain a constant antidiuretic effect without hyponatremia or escape. In case of partial central diabetes insipidus, a non-hormone treatment using compounds which increase vasopressin release or its effect on the kidney can be proposed.

Interleukin 10: a logical candidate for suppressing glomerular inflammation?

Inflammatory processes within the glomerulus are switched off by the local generation of anti-inflammatory mediators. These mediators include eicosanoids (e.g., lipoxins), anti-inflammatory cytokines (interleukins 4 and 13), antagonists of proinflammatory cytokines (interleukin 1 receptor antagonist), neuropoietic cytokines (leukemia inhibitory factor and interleukin 6), as well as deactivators of inflammatory macrophages (transforming growth factor beta and interleukin 10). They limit the effects of proinflammatory mediators by inhibiting their production, stability, or function. Recent attempts to reduce inflammatory lesions in experimental glomerulonephritis have focused on upregulating the expression of these anti-inflammatory mediators by using protein or gene transfer. In particular administration of interleukin 4, interleukin 1 receptor antagonist, leukemia inhibitory factor, or interleukin 10 has been shown to be effective in the treatment of nephrotoxic nephritis. Of all the mediators already tested, interleukin 10 has the greatest potential because of its strong anti-inflammatory effects and weak adverse effects.

Low environmental pH is responsible for the induction of nitric-oxide synthase in macrophages. Evidence for involvement of nuclear factor-kappaB activation.

Stimulation of macrophages with endotoxin and/or cytokines is responsible for the expression of the inducible isoform of nitric oxide synthase (iNOS). Because macrophages are exposed to low pH within the microenvironment of inflammatory lesions, the potential role of acidic pH as an additional regulator of iNOS was investigated. Substitution of the culture medium of rat peritoneal macrophages at pH 7.4 with medium at pH 7.0 up-regulated iNOS activity, as reflected by a 2.5-fold increase in nitrite accumulation. The increase in iNOS activity was associated with a similar increase in iNOS mRNA expression that reflected an increase in iNOS mRNA synthesis rather than stability. Low environmental pH-induced iNOS gene transcription involved the activation of nuclear factor-kappaB (NF-kappaB) transcription factor since exposure of macrophages to low environmental pH both increased NF-kappaB binding activity in the nucleus and enhanced NF-kappaB-driven reporter gene expression. In addition, treatment of macrophages with pyrrolidine dithiocarbamate or n-acetyl-leucinyl-leucinyl-norleucinal, two drugs preventing NF-kappaB translocation to the nucleus, canceled low pH-induced nitrite accumulation. The overall mechanism required the synthesis of tumor necrosis factor alpha (TNFalpha). Indeed, 1) elevated TNFalpha bioactivity was observed in the medium of macrophages exposed to pH 7.0, and 2) incubation of macrophages with a neutralizing anti-TNFalpha antibody impaired both NF-kappaB activation and nitrite accumulation in response to acid challenge. In summary, exposure of macrophages to acidic microenvironment in inflammatory lesions leads to the up-regulation of iNOS activity through the activation of NF-kappaB.

Neutrophil alveolitis in bronchioloalveolar carcinoma: induction by tumor-derived interleukin-8 and relation to clinical outcome.

Tumor infiltrate, predominantly constituted by lymphocytes, may represent an important prognostic factor in bronchioloalveolar carcinoma (BAC), in addition to tumor extension and histological type. In the present study, we determined the presence, the origin, and the prognostic importance of neutrophils that also participate in leukocyte infiltrates of BAC. Neutrophil alveolitis was determined immunohistochemically in both lung biopsies and bronchoalveolar lavage (BAL) fluid samples from 29 patients with histologically proved BAC. The local expression of interleukin (IL)-8 was determined by immunohistochemical and immunoenzymatic techniques. Neutrophil counts were analyzed in relation to the clinical outcome of patients by the Kaplan-Meier method and Cox's univariate and stepwise multivariate models. Lymphocytes and neutrophils dominated the inflammatory cell population in the lower respiratory tract of patients with BAC. Neutrophils were located mainly in the alveolar lumen and seldom in alveolar wall whereas lymphocytes were exclusively present in alveolar wall. A relationship was observed between the number of neutrophils and the level of IL-8 in BAL fluid suggesting the involvement of that chemokine in neutrophil recruitment. The tumor cells were the predominant cells that appeared to express IL-8 by immunolocalization. The presence of increased numbers of neutrophils was significantly associated with a poorer outcome in patients with BAC (P = 0.02). In a multivariate analysis, the neutrophil percentage in BAL fluid was an independent predictor of clinical outcome. The risk of death was increased substantially (rate ratio, 5.2; 95% confidence interval, 1.1 to 24.7) among patients with BAL neutrophil percentage of > or = 39% (median of the distribution) as compared with the others. In BAC, neutrophils accumulate in the alveolar lumen. Elaboration of IL-8 by tumor cells may be responsible for this event, which is associated with a significantly higher risk of death.

Insulin-like growth factors and their binding proteins in pleural fluid.

We investigated the expression and potential regulatory role of insulin-like growth factors (IGFs) and their specific binding proteins (BPs) in tuberculous and nontuberculous pleuritis. By using a radioimmunoassay after acid gel filtration chromatography, we found that mean concentrations of IGF-I were 211.9 +/- 20.2 microg/l and 203.2 +/- 31.1 microg/l in pleural fluid of 14 patients with tuberculous pleuritis and 9 patients with malignant pleuritis respectively. These values were near those in serum of the same patients (221.3 +/- 19.5 microg/l and 204.6 +/- 21.0 microg/l respectively). By using a specific protein-binding assay, we found that mean concentrations of IGF-II were 345.3 +/- 61.0 microg/l and 167.6 +/- 22.7 microg/l in tuberculous and malignant pleural effusions respectively. These values were significantly lower than those in serum of the same patients (628.3 +/- 79.0 microg/l, P<0.025 and 532.0 +/- 85.9 microg/l, P<0.025 respectively). Because bioavailability and bioactivity of IGFs may be regulated by their binding to IGFBPs, we studied IGFBP patterns in the pleural fluid of 6 patients with tuberculous pleuritis. As assessed by Western ligand blotting the levels of IGFBP-1 and IGFBP-2 were increased whereas those of IGFBP-3 were decreased in pleural fluid in comparison with serum. The decrease in IGFPB-3 levels reflected increased proteolysis, as assessed by Western immunoblotting. In spite of this presence of IGFBPs, IGFs could be responsible for the local biosynthesis of 1.25-dihydroxyvitamin D (1,25-(OH)2D) since pleural fluid levels of both IGF-I and IGF-II significantly correlated with those of 1,25-(OH)2D. These results indicate that IGFs are detectable in pleural fluid and may contribute to control the activity of 25-hydroxyvitamin D-1alpha hydroxylase in tuberculous pleuritis.

[Low extracellular pH has a role in the induction of NO synthase type 2 in macrophages]. / La baisse du pH extracellulaire participe à i'induction de la NO synthase de type 2 dans les macrophages.

Stimulation of macrophages with endotoxin and/or cytokines is responsible for the expression of the inducible isoform of nitric oxide synthase (iNOS). Because macrophages are exposed to low pH within the microenvironment of inflammatory lesions, the potential role of low pH as an additional regulator of iNOS was investigated. Substitution of the culture medium of rat peritoneal macrophages at pH 7.4 with medium at pH 7.0 upregulated iNOS activity, as reflected by a 2.5-fold increase in nitrite accumulation. The increase in iNOS activity was associated with a similar increase in iNOS mRNA expression. Low environmental pH-induced iNOS gene expression involved the activation of nuclear factor-kappa B (NF-kappa B) transcription factor since [1] exposure of macrophages to low environmental pH increased NF-kappa B binding activity in the nucleus, and [2] treatment of macrophages with pyrrolidine dithiocarbamate or n-acetyl-leucinyl-norleucinal, two drugs preventing NF-kappa B translocation to the nucleus, canceled low pH-induced nitrite accumulation. The overall mechanism required the synthesis of tumor necrosis factor-alpha (TNF-alpha). Indeed, [1] elevated TNF-alpha bioactivity was observed in the medium of macrophages exposed to pH 7.0, and [2] incubation of macrophages with a neutralizing anti-TNF-alpha antibody impaired both NF-kappa B activation and nitrite accumulation in response to acid challenge. In summary, exposure of macrophages to acidic microenvironment in inflammatory lesions leads to the upregulation of iNOS activity through the activation of NF-kappa B.

Regulation of interleukin-10 production by beta-adrenergic agonists.

Catecholamines have been shown to inhibit some aspects of macrophage activation through a beta receptor-dependent mechanism. This study was undertaken to analyze the effects of isoproterenol, a specific beta-adrenergic agonist, on the synthesis of interleukin-10 (IL-10), a major macrophage-deactivating factor. Isoproterenol increased IL-10 release from lipopolysaccharide-(LPS)-activated mouse peritoneal macrophages in a dose-dependent manner. A significant effect was already observed with 1 microM isoproterenol, while a 4.5-fold increase was achieved with 10 microM. This increase was observed only if macrophages were exposed to isoproterenol for at least 2 h before LPS challenge. It was apparent within 0.5 h and persisted through 24 h at all the LPS concentrations used. A similar increase was observed at the IL-10 mRNA level, as judged by enzyme-linked immunosorbent assay-polymerase chain reaction. The macrophage response to isoproterenol that led to cyclic AMP accumulation was markedly inhibited by H-89, a potent inhibitor of protein kinase A. These data suggest the involvement of cyclic AMP in the regulation of IL-10 synthesis by isoproterenol. IL-10 was in turn partly responsible for a reduction in tumor necrosis factor-alpha synthesis. In vivo, the administration of oxprenolol, a beta-receptor antagonist, significantly reduced serum IL-10 levels 90 min after LPS challenge. Thus, the present study provides the first evidence that endogenous catecholamines are of critical importance in determining the magnitude of the IL-10 response in experimental endotoxemia.