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Proximal tubule cells stimulated by lipopolysaccharide inhibit macrophage activation. Background. Tubule cells can produce a variety of cytokines, extracellular matrix (ECM) components, and adhesion molecules in vitro and in vivo. It is generally assumed that stimulated tubule cells are proinflammatory and at least partially responsible for interstitial inflammation. However, the overall effect of tubular cells on interstitial cells is unknown. In this study, pro- and anti-inflammatory cytokine production and net effects on macrophages of tubule cells activated by lipopolysaccharide (LPS) were examined. Methods. Tubule cells stimulated with LPS expressed tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-12, monocyte chemoattractant protein-1 (MCP-1), IL-10, and transforming growth factor-β (TGF-β). Conditioned media werecollected from confluent monolayers of rat tubule cells stimulated, or not, by LPS for 4 and 18 hours, respectively. Macrophages were cultured with conditioned media and/or LPS (0.5 μg/mL) for 18 hours. Results. TNF-α and IL-lβ mRNA of macrophages stimulated by LPS increased more than fivefold when cultured with control conditioned media from unstimulated tubule cells. Surprisingly, TNF-α and IL-lβ levels of macrophages stimulated by LPS were not increased when cultured with conditioned media from activated tubule cells. Neutralizing antibodies to IL-10 and TGF-β were used to define the inhibitory component(s) in conditioned medium. Anti-IL-10, but not anti-TGF-β, abolished partially the inhibitory effects of conditioned media on macrophages. Conclusion. Tubule cells produce both pro- and anti-inflammatory cytokines and the net effect, partially explained by IL-10, of tubule cells activated with LPS is to inhibit activity of macrophages. Thus, the net effect of activated tubule cells on interstitial pathology may in certain circumstances, be anti- rather than pro-inflammatory. [ABSTRACT FROM AUTHOR]

Background: Since the Fas/Fas Ligand (FasL) interaction is recognized as a major pathway of apoptosis in immune cells, we hypothesized that selective expression of FasL by tubular cells (TC) may promote the resolution of interstitial inflammation by inducing apoptosis of infiltrating immune cells. In this study, the effect of FasL transfection of rat TC on apoptosis of leukocytes was examined.Methods: Rat tubule cells (NRK52E) were transfected with plasmids constructed using human and rat FasL (hFasL and rFasL). The propensity of activated, transfected TC to undergo apoptosis was examined. Similarly, the effects of FasL transfection on apoptosis of Jurkat cells and activated leukocytes were assessed directly following co-culture for 12 hours and in a cell insert system intended to assess the effects of soluble FasL. Fas and FasL expression was assessed by flow cytometry and apoptosis was examined using Annexin V staining and the TUNEL method.Results: Expression of FasL in TC was increased after FasL transfection. Transfected TC showed no detectable increase in apoptosis following lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) activation. Jurkat cell apoptosis was increased ninefold and eightfold after co-culture with TC transfected with hFasL and rFasL, respectively (67.0 +/- 12.1% and 60.1 +/- 8.8% vs. 6.7 +/- 1.8% with un-transfected TC, P < 0.01). Similarly, apoptosis of activated leukocytes was increased fourfold by co-culture (26.8 +/- 4.9% vs. 6.7 +/- 2.0% with untransfected TC, P < 0.01). Leukocyte apoptosis also was increased in an insert culture system (18.2 +/- 4.4% vs. 5.8 +/- 2.3% with un- transfected TC, P < 0.01). No increase of TC apoptosis was detected in any of the co-culture experiments.Conclusion: Enhanced expression of FasL by TC is capable of inducing apoptosis of activated leukocytes, without evidence for increased susceptibility to apoptosis of the transfected cells themselves. This suggests a potential role for this approach in the limitation and resolution of renal tubulointerstitial inflammation. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelial cells is nuclear factor-κB dependent. Background. Endotoxin is an important factor in the development of acute renal failure related to infection and in acceleration of chronic nephritis. Lipopolysaccharide (LPS; the major component of endotoxin) is one of the most potent triggers for renal cells to produce monocyte chemoattractant protein-1 (MCP-1), a key cytokine involved in immune cell recruitment into the renal interstitium in acute and chronic renal diseases. Knowledge about the transcriptional regulation of MCP-1 in renal tubular epithelial cells in response to LPS is incomplete. Methods. Transcriptional regulation of MCP-1 was investigated in rat proximal tubule cells (PTCs) in primary culture and was exposed to LPS using electromobility shift assay and supershift analysis for nuclear factor-κB (NF-κB) and Western blot for the NF-κB inhibitory protein IκB. To prove the role for NF-κB, activator protein (AP-1), and sequence-specific transcription factor (Sp1), mutation and deletion analysis was performed using a 3.5 kb fragment of rat MCP-1 5′-flanking region inserted into a luciferase reporter construct transfected into tubular epithelial cell line (NRK-52E). Results. LPS increased NF-κB in a dose- and time-dependent manner, which paralleled that of MCP-1 mRNA expression. IκBα decreased within 30 minutes of LPS treatment, but returned to basal levels by two hours. IκBβ levels were depressed within one hour and remained low throughout the culture period after LPS stimulation. The activity of the transfected 5′-flanking region of the MCP-1 gene increased nearly threefold after LPS stimulation. Mutation or deletion of NF-κB binding sites, located in the enhancer region of the 5′-flanking region, resulted in a total loss of LPS-induced increase in luciferase activity. Mutation of putative AP-1 and Sp1... [ABSTRACT FROM AUTHOR]

Inhibition of nuclear factor-κB activation reduces cortical tubulointerstitial injury in proteinuric rats. Background.Protein-induced chemokine expression in proximal tubular cells is mediated by the transcription factor nuclear factor-kappa B (NF-κB). We hypothesized that in vivo inhibition of renal NF-κB activation would reduce interstitial monocyte infiltration in a rat model of nonimmune proteinuric tubulointerstitial inflammation. Methods.Male Wistar rats received a single intravenous injection of doxorubicin hydrochloride [adriamycin (ADR), 7.5 mg/kg] and were studied 7, 14, 21, and 28 days later. In a second study, inhibitors of NF-κB [N-acetylcysteine (NAC; 150 mg/kg, b.i.d., i.p.), pyrrolidine dithiocarbamate (PDTC, 50 mg/kg, b.i.d., i.p.)] or vehicle were commenced on day 14 after the onset of proteinuria and were continued until day 30. Results.Rats injected with ADR had increased proteinuria (UpV, day 28, 474 ± 57; control, 18 ± 2 mg/day; P < 0.01) and cortical tubulointerstitial injury [tubule cell atrophy, interstitial volume, and monocyte/macrophage (ED-1) infiltration]. Electrophoretic mobility shift assay of nuclear extracts from whole cortex of ADR rats demonstrated that NF-κB activation (p50/65, p50/c-Rel) increased from day 7 (4.7 ± 0.2 fold-increase above control; P < 0.01) was maximal at day 28 (6.2 ± 0.7; P < 0.01) and correlated with UpV (r = 0.63; P < 0.05) and interstitial ED-1 infiltration (r = 0.67; P < 0.01). Chronic treatment of ADR rats with PDTC suppressed NF-κB activation (by 73%; P < 0.05) without any effect on UpV. NF-κB inhibition with PDTC was accompanied by a reduction in tubule cell atrophy (59%; P < 0.01), interstitial volume (49%; P < 0.05) and ED-1 infiltration (48%; P < 0.01), and cortical lipid peroxidation (41%; P < 0.05) compared with vehicle-treated ADR rats. In contrast NAC had no effect on NF-κB activation,... [ABSTRACT FROM AUTHOR]