Expression and characterization of recombinant rat alpha 3(IV)NC1 and its use in induction of experimental autoimmune glomerulonephritis.

BACKGROUND: The autoantigen in Goodpasture's disease is known to be the non-collagenous domain of the alpha3 chain of type IV collagen, alpha 3(IV)NC1. There is mounting evidence that alpha 3(IV)NC1 is also a target of autoimmunity in experimental autoimmune glomerulonephritis (EAG). Sado et al. [Kidney Int 1998; 53, 664-671] have reported that recombinant human alpha 3(IV)NC1 and alpha4(IV)NC1 are nephritogenic in WKY rats. We have proposed that immunization with homologous antigen is more appropriate for detailed investigation of autoimmunity in EAG. METHODS: To this end, we have cloned and sequenced rat alpha 3(IV)NC1 and expressed it in COS-7 cells. Recombinant rat alpha 3(IV)NC1, secreted into the COS-7 cell supernatant, was purified on an anti-M2 FLAG affinity column and characterized by western blotting. Recombinant antigen was then used to immunize WKY rats, in order to induce EAG. RESULTS: The recombinant material was antigenic as judged by binding to sera from patients with Goodpasture's disease and a mAb to alpha 3(IV)NC1. Immunization of WKY rats (n=5), with recombinant rat alpha 3(IV)NC1 in FCA at a dose of 1 mg/kg resulted in circulating anti-GBM antibodies directed towards alpha 3(IV)NC1, linear deposits of IgG on the GBM, albuminuria, deposits of fibrin in the glomeruli, severe focal necrotizing glomerulonephritis with crescent formation, and glomerular influx of CD8+ T cells and macrophages. Western blot analysis demonstrated that sera from these rats bound strongly to recombinant rat alpha 3(IV)NC1, as well as to collagenase-solubilized human and rat GBM. The pattern of binding was indistinguishable from that of sera from patients with Goodpasture's disease. CONCLUSIONS: This purified recombinant rat alpha 3(IV)NC1, which is both antigenic and nephritogenic, will be of value in analysing autoimmune responses in experimental anti-GBM disease.     

Cytoskeletal protein expression and regenerative markers in schistosomal nephropathy.

BACKGROUND: Progression of renal diseases is related to the abnormal regulation of cellular and extracellular matrix turnover. Other factors in addition to schistosomal antigens may be relevant to the progression of schistosomal nephropathy (SN). The validity of markers of fibroblastic differentiation, alpha smooth muscle actin (alphaSMA), and vimentin, as well as the regenerative activity (PCNA/apoptosis index) in determination of progression of SN in comparison to other forms of non-schistosomal nephropathy (non-SN) is investigated. METHODS: Three groups were included; group I pure SN (n=16), group II a diverse group of non-schistosomal patients with comparable pathologic changes on renal biopsy (n=40) and a control group (n=5). Immunohistochemical staining of myofibroblasts (alphaSMA and vimentin) and proliferating cells (PCNA) and histomorphometric analysis was done. In situ end labelling (ISEL) of DNA was used to evaluate apoptosis. RESULTS: No differences in the patterns of distribution of positivity of the different studied markers were observed between the different nephropathy groups. Both alphaSMA and vimentin were detected in glomerular mesangial, tubular epithelial, interstitial inflammatory fibroblast-like cells and occasionally endothelial cells. PCNA and apoptotic cells were detected in tubular epithelial and interstitial cells with paucity of positive cells in the glomerulus. Significant positive correlations were detected in group I between glomerular sclerosis and interstitial markers including interstitial alphaSMA (r=0.609, P=0.001), interstitial vimentin (r=0.812, P=0.00) and interstitial apoptosis (r=0.733, P=0.001). On the other hand, glomerulosclerosis in group II showed significant positive correlations with predominantly the glomerular markers; glomerular alphaSMA (r=0.475, P=0.002), glomerular apoptosis (r=0.684, P=0.00) and glomerular PCNA (r=0.691, P=0.00). Interstitial fibrosis correlated significantly with interstitial markers in group I including interstitial alphaSMA (r=0.837, P=0.00) interstitial vimentin (r=0.929, P=0.00), interstitial apoptosis (r=0.807, P=0.00) and interstitial PCNA (r=0.617, P=0.01), while in group II it correlated with both interstitial and glomerular markers. In addition, the tubulo-interstitial ratio was significantly higher in group I in comparison with group II (P=0.024), with no difference between groups II and III. CONCLUSIONS: Although SN may start as glomerulopathy associated with increased mesangial cellularity, the interstitial rather than the glomerular markers of myofibroblastic differentiation and those of cell turnover are playing a crucial role in late stages of schistosomal, but not in non-schistosomal nephropathies.     

Hypoxia reduces the expression and anti-inflammatory effects of peroxisome proliferator-activated receptor-gamma in human proximal renal tubular cells.

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-gamma may counteract tissue fibrosis via its anti-inflammatory actions, while hypoxia, a new pro-fibrotic factor, reportedly modifies PPAR-gamma expression. However, the effects of hypoxia on the expression and anti-inflammatory actions of PPAR-gamma have yet remained to be clarified in renal tubular cells. METHODS: Confluent human proximal renal tubular epithelial cells (HPTECs) were exposed to hypoxia (1% O2) and/or TNF-alpha at 10 ng/ml for up to 48 h. The cells were incubated with PPAR-gamma agonists, 15d-PGJ2 or pioglitazone, for 30 min before stimulation. Precise amounts of PPAR-gamma and MCP-1 mRNA and protein were measured by TaqMan quantitative PCR and immunoblot or ELISA, respectively. RESULTS: A cDNA array analysis identified PPAR-gamma as one of the hypoxia-affected genes in HPTECs. Hypoxia reduced mRNA levels of PPAR-gamma at 24 and 48 h and protein levels at 6 and 48 h. Knockout of hypoxia-inducible factor-1alpha (HIF-1alpha) with its dominant negative form did not block the hypoxia-induced reduction in PPAR-gamma expression. PPAR-gamma's activation with 15d-PGJ2 or pioglitazone reduced basal and TNF-alpha-stimulated MCP-1 expression at mRNA and protein levels at 24 h under normoxia. MCP-1 reduction rates at basal mRNA and protein levels were slightly but significantly lower during hypoxia than normoxia (9 vs 69% and 36 vs 42%, respectively, for 15d-PGJ2, and 0 vs 34% and 12 vs 21%, respectively, for pioglitazone). Finally, a specific inhibitor for PPAR-gamma, GW9662, weakened the MCP-1-decreasing effect of 15d-PGJ2 by about 30%, under basal conditions, while it abolished the effect of pioglitazone almost completely. CONCLUSIONS: Hypoxia-induced loss of function of PPAR-gamma reduces anti-inflammatory effects of PPAR-gamma activation, possibly modulating inflammatory responses in the diseased kidney.     

p38 MAPK phosphorylation and NF-kappa B activation in human crescentic glomerulonephritis.

BACKGROUND: p38 mitogen-activated protein kinase (p38 MAPK) followed by the activation of NF-kappa B participates in the intracellular signal transduction and production of cytokines and chemokines. The pathophysiological roles of p38 MAPK and NF-kappa B in human glomerulonephritis, however, remain to be investigated. METHODS: We investigated the phosphorylated p38 MAPK (p-p38 MAPK) and activated NF-kappa B immunohistochemically in the kidneys of 34 patients with crescentic glomerulonephritis and 26 control patients with thin basement membrane disease and minimal change nephrotic syndrome. We also explored the co-localization of p-p38 MAPK with CCR5, the signal of which leads to p38 MAPK activation. Furthermore, urinary levels of MIP-1 alpha, the cognate ligand for CCR5, were determined by enzyme-linked immunosorbent assay. RESULTS: p-p38 MAPK-positive cells and activated NF-kappa B-positive cells were mainly detected in crescentic lesions, tubular epithelial cells, and interstitial mononuclear infiltrates. The number of p-p38 MAPK-positive cells in patients with crescentic glomerulonephritis was higher than that in control patients. The number of p-p38 MAPK-positive cells in glomeruli was well correlated with the percentage of cellular crescents, the number of CD68-positive cells, and urinary MIP-1 alpha levels. In addition, the number of activated NF-kappa B-positive cells was well correlated with the number of p-p38 MAPK-positive cells in glomeruli. Dual staining revealed that most of CCR5-positive cells were positive for p-p38 MAPK. Finally, p-p38 MAPK-positive cells and activated NF-kappa B-positive cells decreased during glucocorticoid therapy-induced convalescence. CONCLUSIONS: We conclude that the phosphorylation of p38 MAPK associated with the activation of NF-kappa B may be involved in the upregulation of intrarenal MIP-1 alpha and the utilization of CCR5 signalling, which may result in human crescentic glomerulonephritis.     

Platelet-derived growth factor, basic fibroblast growth factor, and interferon gamma increase type IV collagen production in human fetal mesangial cells via a transforming growth factor-beta-dependent mechanism.

BACKGROUND: Glomerulosclerosis is characterized by glomerular accumulation of extracellular matrix following mesangial cell proliferation. The precise pathomechanism of glomerulosclerosis is still undetermined. Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (b-FGF) are known to be mitogenic for mesangial cells, and interferon gamma (IFN-gamma) is known to have an inhibitory effect on mesangial cell proliferation. We attempted to clarify the role of these cytokines on mesangial matrix production using cultured human fetal mesangial cells (HMC). METHODS: HMC were incubated with these cytokines for 24-72 h and the levels of type IV collagen and TGF-beta in the cell supernatants were measured by enzyme immunoassay. RESULTS: PDGF, b-FGF, and IFN-gamma stimulated type IV collagen production by HMC in a dose- and time-dependent manner. The anti-TGF-beta neutralizing antibody clearly inhibited their stimulatory effect on type IV collagen production. PDGF and b-FGF also stimulated TGF-beta production by HMC in a dose-dependent manner, although IFN-gamma did not. CONCLUSION: PDGF, b-FGF, and IFN-gamma stimulate type IV collagen production in cultured HMC via a TGF-beta-dependent mechanism.     

Alphav integrin regulates TNF-alpha-induced nitric oxide synthesis in rat mesangial cells--possible role of osteopontin.

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) induces nitric oxide (NO) synthesis in rat mesangial cells (MCs). We previously demonstrated that osteopontin (OP), a matrix protein that mainly interacts with the alphav integrin family, increased time-dependently by TNF-alpha stimulation at gene and protein levels. The regulation of NO synthesis by integrins or matrix proteins is unclear. METHODS: We examined whether integrin, especially alphav integrin, regulates NO synthesis in rat MCs and whether OP, an alphav integrin ligand, has an effect on TNF-alpha-induced NO synthesis. Furthermore, OP and inducible NO synthase (iNOS) gene expression was examined by Northern blotting. RESULTS: TNF-alpha increased NO synthesis in MCs in a time-dependent manner. Synthetic GRGDSP peptide, which is known to inhibit various integrins that interact with RGD-containing extracellular matrices, increased TNF-alpha-induced NO levels in a dose-dependent manner. Cyclical RGD peptide, the specific inhibitor of alphav integrin, also exhibited a dose-dependent effect of increasing NO levels, while GRGESP peptide, which has very low affinity to integrins, had no effect. In addition, NO synthesis was found to be significantly reduced when MCs were plated on OP-coated dishes compared to type I or IV collagen-coated dishes. Furthermore, anti-OP antibody increased NO synthesis in MCs. iNOS mRNA levels were increased by TNF-alpha, and were abruptly diminished after OP mRNA was significantly induced. CONCLUSIONS: The present study demonstrated the involvement of alphav integrin in TNF-alpha-induced NO synthesis in rat MCs, and the possible role of OP was suggested in the mechanism. TNF-alpha and extracellular matrices can co-operate to regulate the behaviour of MCs at least partly through NO synthesis, which may participate in the course of glomerular diseases.     

Rosiglitazone ameliorates cisplatin-induced renal injury in mice.

BACKGROUND: Inflammatory mechanisms may play an important role in the pathogenesis of cisplatin nephrotoxicity. Agonists of the peroxisome proliferator-activated receptor-gamma (PPARgamma), such as rosiglitazone, have been recently demonstrated to regulate inflammation by modulating the production of inflammatory mediators and adhesion molecules. The purpose of this study was to examine the protective effects of rosiglitazone on cisplatin nephrotoxicity and to explore the mechanism of its renoprotection. METHODS: Mice were treated with cisplatin with or without pre-treatment with rosiglitazone. Renal functions, histological findings, aquaporin 2 (AQP2) and adhesion molecule expression, macrophage infiltration and tumour necrosis factor-alpha (TNF-alpha) levels were investigated. The effect of rosiglitazone on nuclear factor (NF)-kappaB activity and on viability was examined using cultured human kidney (HK-2) cells. RESULTS: Rosiglitazone significantly decreased both the damage to renal function and histological pathology after cisplatin injection. Pre-treatment with rosiglitazone reduced the systemic levels of TNF-alpha and down-regulated adhesion molecule expression in addition to the infiltration of inflammatory cells after cisplatin administration. Rosiglitazone restored the decreased AQP2 expression after cisplatin treatment. Pre-treatment with rosiglitazone blocked the phosphorylation of the p65 subunit of NF-kappaB in cultured HK-2 cells. Rosiglitazone had a protective effect via a PPARgamma-dependent pathway in cisplatin-treated HK-2 cells. CONCLUSION: These results showed that pre-treatment with rosiglitazone attenuates cisplatin-induced renal damage through the suppression of TNF-alpha overproduction and NF-kappaB activation.     

Inflammatory mediators in human renal dysplasia.

BACKGROUND: Cytokines regulate many processes in the immune system and have recently been implicated in normal organogenesis. We previously demonstrated that the archetypal inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) is expressed in the murine metanephros, and exogenous TNF-alpha inhibits nephrogenesis and increases macrophage numbers in vitro (Cale et al., Int J Dev Biol 1998; 42: 663-674). The phenotype seen, with an arrest of ureteric bud branching and failure of mesenchymal to epithelial conversion, is similar to human renal dysplasia. Methods and results. In normal human fetal kidneys we demonstrated the presence of macrophages and T cells and also documented TNF receptor expression on ureteric bud derivatives. In contrast to normal tissues, TNF-alpha protein was detected in dysplastic kidneys. This factor was also detected in the urine of fetuses with obstructive uropathy and TNF receptors were expressed in dysplastic tubules. Furthermore, we noted a fetal distribution of macrophages and T cells in dysplastic tissues and persistent expression of the adhesion molecules neural cell adhesion molecule and intercellular adhesion molecule. CONCLUSIONS: We suggest that abnormal expression of cytokines early in renal development dysregulates normal patterns of adhesion molecule expression and inflammatory cells, and may contribute to the pathogenesis of renal dysplasia.     

Interstitial expression of alpha-SMA: an early marker of chronic renal allograft dysfunction.

BACKGROUND: Renal myofibroblast infiltration has been shown to be strongly associated with renal function decline in several chronic renal diseases. The purpose of the present study was to investigate whether early detection of myofibroblast infiltration using alpha-smooth-muscle actin (alpha-SMA) expression in time-zero biopsies predicts renal allograft dysfunction. METHODS: We studied renal tissue from 38 renal transplant patients from whom biopsies had been taken after vascular anastomosis during transplantation to ascertain whether myofibroblasts infiltration predicts renal graft survival. Immunohistochemistry was performed on time-zero biopsies to determine alpha-SMA expression, and this was compared to annual glomerular filtration rate (GFR) variation and other parameters including cold ischaemic time (CIT), donor and recipient age, number of acute rejections, and delayed graft function (DGF). GFR was measured by inulin clearance during of 3 years of follow-up after the transplantation. Progressors were defined as patients with an annual GFR decline >5 ml/min/year. RESULTS: We found a significant correlation between interstitial alpha-SMA expression in time-zero biopsies and GFR evolution during the post-transplantation course (r=0.60, P<0.001). Although progressors had greater interstitial alpha-SMA expression than non progressors (7.9+/-0.7 vs 4.3+/-0.4%), they showed only a tendency towards higher glomerular alpha-SMA expression. In addition, progressors had more interstitial fibrosis in time-zero biopsies than non-progressors. There was no relationship between alpha-SMA expression and CIT, donor and recipient ages, number of acute rejections, and occurrence of DGF. CONCLUSION: This study suggests that alpha-SMA evaluation in time-zero biopsies, especially the combination of alpha-SMA expression and interstitial fibrosis, can strongly predict chronic renal allograft dysfunctions.     

Morphology and distribution of chandelier cell axon terminals in the mouse cerebral cortex and claustroamygdaloid complex

Chandelier cells represent a unique type of cortical --aminobutityricacidergic interneuron whose axon terminals (Ch-terminals) onlyform synapses with the axon initial segments of some pyramidalcells. Here, we have used immunocytochemistry for the high-affinityplasma membrane transporter GAT-1 and the calcium-binding proteinparvalbumin to analyze the morphology and distribution of Ch-terminalsin the mouse cerebral cortex and claustroamygdaloid complex.In general, 2 types of Ch-terminals were distinguished on thebasis of their size and the density of the axonal boutons thatmade up the terminal. Simple Ch-terminals were made up of 1or 2 rows of labeled boutons, each row consisting of only 3–5boutons. In contrast, complex Ch-terminals were tight cylinder-likestructures made up of multiple rows of boutons. Simple Ch-terminalswere detected throughout the cerebral cortex and claustroamygdaloidcomplex, the complex type was only occasionally found in certainregions, whereas in others they were very abundant. These resultsindicate that there are substantial differences in the morphologyand distribution of Ch-terminals between different areas andlayers of the mouse cerebral cortex. Furthermore, we suggestthat the distribution of complex Ch-terminals may be relatedto the developmental origin of the different brain regions analyzed.     

Interleukin 6 production by human proximal tubular epithelial cells in vitro: analysis of the effects of interleukin-1{alpha} (IL-1{alpha}) and other cytokines

Proximal tubular epithelial cells (PTEC) from human renal tissueobtained from biopsy or nephrectomy were grown in monocultureand evaluated in vitro at passage 2–4 for interleukin6 (IL-6) production in response to medium alone or to interleukin1 alpha (IL-1), tumour necrosis factor alpha (TNF), interleukin2 (IL-2), interferon gamma (INF) or lipopolysaccharide (LPS).IL-6 bioactivity was quantitated using the IL-6-dependent murinehybridoma cell line (B9) and expressed as IL-6 units/ml/10⁵PTEC. PTEC cell lines exposed to medium alone produced intermediateamounts of IL-6 with substantial variability between cell lines.Introduction of IL-1 resulted in a dose- and time-dependentincrease in IL-6 production by PTEC that was maximal at 1 ng/mlIL-1 at 24 h. All PTEC cell lines showed an increased IL-6 productionon exposure to IL-1 varying from 1.3- to 24-fold increase overbaseline production. This response was completely blocked byanti-rIL-1. No significant IL-6 production by PTEC could beinduced by TNF, IL-2, IFN, or LPS over a broad dosage range.Cycloheximide inhibited IL-6 production without irreversiblecell toxicity, indicating de-novo synthesis. IL-6 produced byPTEC had a molecular weight of 26-29 kDa as demonstrated byWestern blot analysis. Using PCR analysis we could demonstrateupregulation by IL-1 of IL-6 mRNA in a dose-response fashion,indicating that IL-1 regulates IL-6 production at a pretranslationalvalue of protein synthesis. These results show that human culturedPTEC produce IL-6 under both normal and IL-1-stimulated conditions,and suggest that they may have a regulatory function in responseto cytokines in the setting of inflammation in the renal cortex.     

Plasma concentrations of alpha-melanocyte-stimulating hormone are elevated in patients on chronic haemodialysis.

BACKGROUND: Clinical and/or laboratory signs of systemic inflammation occur frequently in patients undergoing long-term haemodialysis. It is likely, therefore, that a compensatory release of endogenous anti-inflammatory molecules occurs to limit host reactions. The aim of the present research was to determine if the potent anti-inflammatory peptide alpha-melanocyte-stimulating hormone (alpha-MSH), a pro-opiomelanocortin derivative, is increased in plasma of haemodialysis patients. Because endotoxin and cytokines induce alpha-MSH in vivo and in vitro, we also measured plasma concentrations of endotoxin, interleukin-6 (IL-6), and tumour necrosis factor alpha (TNF-alpha), and the two circulating products of activated monocytes, nitric oxide (NO) and neopterin. METHODS: Thirty-five chronic haemodialysis patients, 20 patients with chronic renal failure not yet on dialysis, and 35 normal controls were included in the study. In the haemodialysis group, blood samples were obtained before and at the end of a dialysis session. Plasma alpha-MSH was measured using a double antibody radioimmunoassay, and IL-6, TNF-alpha, and neopterin using specific enzyme-linked immunosorbent assays. Plasma nitrites were determined by a colorimetric method, and endotoxin with the quantitative chromogenic LAL (limulus amoebocyte lysate) method. RESULTS: Mean plasma alpha-MSH was higher in haemodialysis patients than in control subjects, with the peptide concentrations being particularly elevated in dialysed patients with detectable endotoxin. High alpha-MSH concentrations were observed in the pre-dialysis samples, with no substantial change at the end of the dialysis session. Plasma concentrations of IL-6, TNF-alpha, neopterin, and NO were generally elevated in chronic haemodialysis patients and there was a negative correlation between circulating alpha-MSH and IL-6. In patients with renal failure not yet on dialysis, mean plasma alpha-MSH was similar to that of normal subjects. CONCLUSIONS: alpha-MSH is increased in the circulation of chronic haemodialysis patients and particularly so in case of detectable endotoxaemia. Reduction of renal clearance is unlikely to contribute to the observed rise of the peptide because alpha-MSH concentration is not increased in patients with chronic renal failure who are not yet on dialysis. It is likely that dialysis-associated endotoxaemia, directly and/or through cytokine release, enhances the production of the anti-inflammatory mediator alpha-MSH that limits host reactions.     

Myofibroblasts, predictors of progression of mesangial IgA nephropathy?

The limited knowledge of the cellular mediators of renal scarringhampers progress in the management of progressive chronic renalfailure (CRF). We have studied 38 patients with biopsy-provenmesangial IgA nephropathy with emphasis on attempting to definethe role of myofibroblasts(-smooth muscle actin/SMA-positivecells) in renal scarring. In 18 untreated patients, correlationswere undertaken between known histological parameters of progressionas well as the presence of myofibroblasts in tissues and theclinical outcome. -SMA staining by an avidin-biotin-peroxidasemethod was confined to a large extent to the vascular smoothmuscle cells of normal kidneys but extended to the tubulointerstitiumand periglomerular space in scarred kidneys. Mild glomerularstaining was also noted. The interstitial immunostain followeda similar distribution to that of interstitial type III collagen.Morphometric analysis showed the interstitial SMA staining tobe a reliable histological predictor of outcome as it discriminatedbetween progressors and non-progressors (²=4.923, P=0.026).The intensity of the interstitial -SMA staining correlated withrenal functional outcome; inversely with the reciprocal of serumcreatinine slopes (r=-0.466, P<0.025) and positively withthe serum creatinine value at the end of the observation period(r=0.704, P<0.00l). Other histological parameters that correlatedwith outcome included the degree of tubulointerstitial (TI)inflammatory infiltrate (r=-0.425, P<0.05 with 1/Cr slopeand r=0.760, P< with serum creatinine) and the intensityof the TI staining for collagen IV (r=-0.567 and 0.667 respectively).In 20 patients treated with prednisolone and azathioprine, asecond renal biopsy showed the persistence of interstitial myofibroblastsin the absence of progressive fibrosis. In conclusion, stainingof renal biopsies of patients with mesangial IgA for -SMA-positivecells may identify the myofibroblasts as important mediatorsof TI scarring and have useful prognostic implications.     

Pyrrolidine dithiocarbamate exerts anti-proliferative and pro-apoptotic effects in renal cell carcinoma cell lines.

BACKGROUND: The activation of nuclear factor-kappaB (NF-kappaB) has been implicated in the development, progression and metastasis of renal cell carcinoma (RCC). This study investigates the effect of pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, on two metastatic human RCC cell lines, ACHN and SN12K1. METHODS: RCC cell lines and normal cells were exposed to 25 or 50 microM of PDTC. Apoptosis was measured by flow cytometry and TdT-mediated nick end labelling methods. Cell viability and proliferation were measured by MTT and BrdU assays, respectively. Expression of NF-kappaB subunits, IkappaBs, IkappaB Kinase (IKK) complex and apoptotic regulatory proteins were analysed by western blotting and/or immunofluorescence. DNA-binding activity of NF-kappaB subunits were measured by ELISA. RESULTS: RCC cell lines had a higher basal level expression of all the five subunits of NF-kappaB than normal primary cultures of human proximal tubular epithelial cells or HK-2 cells. PDTC decreased the viability and proliferation of RCC, but not normal cells. Of the two RCC cell lines, ACHN had a higher basal level expression of all the five NF-kappaB subunits than SN12K1 and was more resistant to PDTC. While PDTC induced an overall decrease in expression of all the five NF-kappaB subunits in both RCC cell lines, unexpectedly, it increased the nuclear expression of NF-kappaB in ACHN, but not in SN12K1. PDTC reduced the DNA-binding activity of all the NF-kappaB subunits and the expression of the IKK complex (IKK-alpha, IKK-beta and IKK-gamma) and the inhibitory units IkappaB-alpha and IkappaB-beta. PDTC induced a significant increase in apoptosis in both RCC cell lines. This was associated with a decrease in expression of the anti-apoptotic proteins, Bcl-2 and Bcl-(XL), without marked changes in the pro-apoptotic protein Bax. CONCLUSION: These data suggest that PDTC has the potential to be an anticancer agent in some forms of RCC.