Role of tumor-associated macrophages in renal cell carcinoma.

We investigated the biologic meaning of tumor-associated macrophages (TAM) in renal cell carcinoma (RCC). The study group comprised of 83 patients with RCC. TAM was isolated by plastic adherence following enzymatic digestion of surgically removed tumor tissues. In some of the patients, monocytes were also isolated from peripheral blood mononuclear cells by plastic adherence. When TAM and monocytes were compared in the same patients, TAM produced interleukin 6 (IL-6), tumor necrosis factor alpha (TNFalpha) and interleukin 1beta (IL-1beta) without lipopolysaccharide (LPS) stimulation, while monocytes hardly produced IL-6, TNFalpha and IL-1beta without LPS stimulation. However, with LPS stimulation, monocytes produced more IL-6, TNFalpha and IL-1beta than TAM. In stage T1 RCC patients, there was a significant positive correlation between TNFalpha production of TAM and tumor size. In order to investigate the effects of TAM on cancer cells, TAM was co-cultured with A498, K562 and in some cases, with short-term established RCC lines for 96 h. As a result, TAM largely enhanced cell proliferation. These results suggested that TAM may play an important role in certain steps of tumor progression.

Effects of tumor necrosis factor alpha in renal cell carcinoma.

Tumor necrosis factor alpha (TNFalpha) and TNFalpha receptor mRNA expression, the effects of TNFalpha on DNA synthesis and cell proliferation as well as its effects on interleukin-6 (IL-6) production and expression in renal cell carcinoma (RCC) were studied using RCC cell lines. The effects of TNFalpha on DNA synthesis and IL-6 production were also studied using short-term established RCC cell lines. A total of 8 cell lines, 4 RCC cell lines (RC-2, RGB, 14T, 4T) and 4 cell lines established at our laboratory (OCUU-1, 2, 4, 5), as well as 10 short-term established RCC cell lines were used. When TNFalpha and TNFalpha receptor mRNA expression was examined by RT-PCR, p55 TNF receptor mRNA expression was observed while TNFalpha and p75 TNF receptor mRNA expression was not observed in all cell lines. When the effects of TNFalpha on DNA synthesis were studied by [3H]-thymidine incorporation assay, DNA synthesis was induced in RGB, 14T, 4T, OCUU-2 and OCUU-4 by adding 10 and 100 pg/ml of TNFalpha while it was not induced in RC-2 and OCUU-5 at all concentrations. When its effects on cell proliferation were evaluated by MTT assay, cell proliferation was observed in RGB, 14T and 4T at TNFalpha concentration of 10 pg/ml and in RGB, 14T, 4T, OCUU-4 and OCUU-5 at TNFalpha concentration of 100 pg/ml. However, cell proliferation was not detected in RC-2 and OCCU-2 at any concentration. When the effects of TNFalpha on IL-6 production were studied by ELISA, IL-6 production was induced in RC-2, RGB, 14T, OCUU-1, OCUU-2 and OCUU-5 while not in 4T and OCUU-4. When its effects on IL-6 expression were examined by Northern blot analysis, the results were similar to those obtained by ELISA. As for the 10 short-term established RCC cell lines, DNA synthesis and IL-6 production were induced with the addition of TNFalpha. These results suggested that TNFalpha induced cell proliferation in RCC.

Norepinephrine activates P44 and P42 MAPK in human prostate stromal and smooth muscle cells but not in epithelial cells.

BACKGROUND: In vascular smooth muscle cells, alpha1-adrenergic stimulation increases DNA synthesis and cell proliferation via activation of p44/42 (ERK1/2) MAPK. We examined whether norepinephrine (NE) activates MAPK and stimulates the proliferation of prostatic epithelial and non-epithelial cells. METHODS: Human prostatic epithelial cells, stromal cells, and smooth muscle cells were purchased from BioWhittaker (Walkersville, MD). After reaching a semi-confluent condition, the cells were cultured in RPMI-1640 without serum for 1 day. At 10 min after adding NE (10(-6) or 10(-7) M) to the medium, the cells were collected. Cell lysate was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot using anti-phospho-p44/42 and anti-p44/42 antibodies. The activation of p44/42 was estimated by the ratio of phospho-p44/42 to total p44/42. Cell proliferation was evaluated by (3)H-thymidine uptake assay. After reaching a semi-confluent condition, the cells were cultured in RPMI-1640 containing 0.5% FCS with or without NE (10(-6) or 10(-7) M) for 16 hr followed by a (3)H-thymidine uptake period (24 hr). RESULTS: P44/42 MAPK was significantly activated by NE in non-epithelial cells (stromal cells and smooth muscle cells) while not in epithelial cells. The uptake of (3)H-thymidine was significantly increased by NE in both non-epithelial cells, which was inhibited by alpha1-adrenoceptor antagonists. CONCLUSIONS: These results suggest that NE may stimulate the proliferation of non-epithelial prostatic cells, which may be involved in the pathogenesis of BPH.

Echography of left ventricular end-diastolic diameter as a reliable tool for estimating "dry weight" in hemodialysis patients.

PURPOSE: It is hard to decide an accurate value for the so-called dry weight (DW) in hemodialysis patients with cardiac disease. Our objective is to evaluate the efficacy of echocardiography to decide DW. PATIENTS AND METHODS: 115 patients on hemodialysis were divided into 2 groups: the cardiac disease group and non-cardiac disease group. The relationship between atrial natriuretic peptide (ANP) and left ventricular end-diastolic diameter (LVDd) measured by echocardiography was examined. RESULTS: There was a significant positive relationship between ANP and LVDd in the noncardiac disease group, but no significant relationship was noted in the cardiac disease group. The DW was re-evaluated and transferred into more suitable criteria for the patients who often became unconscious with decreased blood pressure during dialysis. When LVDd slightly increased beyond the new criteria of dry weight, unconsciousness was disappeared and blood pressure became stable. CONCLUSIONS: Echocardiography examination is very beneficial for screening to determine DW for the patients with cardiac disease.

Concentration gradient of oxalate from cortex to papilla in rat kidney.

BACKGROUND: The kidney eliminates the major fraction of plasma oxalate. It is well known that oxalate is freely filtered by glomeruli and secreted by the proximal tubules. However, the renal handling of oxalate in distal nephrons, which is considered as playing an important role in stone formation, remains obscure. METHODS: At 15-180 min after intravenous injection of 14C-oxalate to rats, the intrarenal localization of radioactivity was quantitatively measured by the radioluminographic method using a bioimaging analyzer. Tissue radioactivity was compared with plasma, and urinary radioactivities were measured by a liquid scintillation counter. The control study was conducted with 14C-inulin. RESULTS: The radioactivity of 14C-oxalate in the papilla was 10 times greater than in the cortex and eight times greater than in the medulla 180 min after injection when almost no radioactivity was present in the urine. In contrast, the radioactivity of 14C-inulin was nine times less in the papilla than in the cortex at the same time. CONCLUSION: Oxalate remains in the renal papilla for an extended period. This accumulation of oxalate may be attributed to calcium oxalate crystal fixation along the deep nephron which is considered to be the first step of stone formation.

Inhibition of nuclear factor-kappaB activation by pyrrolidine dithiocarbamate prevents chronic FK506 nephropathy.

BACKGROUND: Chronic tacrolimus (FK506) nephrotoxicity is characterized by renal fibrosis with interstitial inflammation. Since nuclear factor-kappaB (NF-kappaB) plays a key role in chronic inflammatory diseases including renal disease, the present study was conducted to elucidate the role of NF-kappaB in the pathogenesis of chronic FK506-induced nephropathy. METHODS: FK506 (1 mg/kg/day, SC) was administered daily to rats maintained on low sodium diet for 42 days. Some rats were treated with a putative NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC; 100, 200 mg/kg/day, by gavage). The renal function, renal histology, renal NF-kappaB-DNA binding activity and gene expression profile were examined. RESULTS: FK506 caused a decline in glomerular filtration and induced characteristic renal morphologic changes including arteriolopathy, tubular atrophy and interstitial fibrosis. FK506 markedly activated renal cortical NF-kappaB-DNA binding. PDTC administration inhibited NF-kappaB-DNA binding activity in a dose dependent manner. With higher dose, NF-kappaB-DNA binding activity was decreased to a control level. PDTC had little effect on FK506-induced renal dysfunction. Renal cortical monocyte/macrophage infiltration observed in FK506-treated rats was dramatically suppressed by PDTC. FK506 up-regulated renal cortical gene expression of chemoattractant proteins, monocyte chemoattractant protein-1 (MCP-1) and osteopontin. PDTC significantly blocked MCP-1 gene expression but had no effect on osteopontin gene expression. Tubular atrophy and tubulointerstitial fibrosis, but not arteriolopathy, were significantly attenuated by PDTC. FK506 increased renal mRNA expression of fibrogenic molecules and extracellular matrices that also were attenuated by PDTC treatment. CONCLUSIONS: NF-kappaB plays an important role in mediating cortical monocyte/macrophage infiltration and in the pathogenesis of tubular injury and interstitial fibrosis in experimental FK506-induced chronic nephropathy.

Magnesium supplementation prevents experimental chronic cyclosporine a nephrotoxicity via renin-angiotensin system independent mechanism.

BACKGROUND: We have previously shown that correction of hypomagnesemia by magnesium (Mg) supplementation ameliorates chronic cyclosporine A (CsA) nephropathy via inhibiting gene expression of fibrogenic molecules. Experiments were conducted to further elucidate upstream mechanism of the beneficial effects upon CsA nephrotoxicity. METHODS: CsA (15 mg/kg/day, subcutaneous [SC]) was administered daily to rats maintained on low sodium diet for 7, 14, and 28 days. Because blockade of renin-angiotensin system improves chronic CsA nephropathy, the effects of Mg supplementation and those of angiotensin-converting enzyme inhibitor (ACEI) were compared on renal function, renal histology, mononuclear cell infiltration, and gene expression profile. RESULTS: CsA induced a decline in glomerular filtration and developed characteristic striped fibrosis that were mostly evident at day 28. Mg attenuated CsA-induced impaired renal function, whereas ACEI did not. Interstitial inflammation as evidenced by monocyte/macrophage infiltration preceded the renal fibrosis and increased progressively with the CsA treatment period. Concomitantly, CsA markedly up-regulated expression of chemoattractant proteins, osteopontin, and monocyte chemoattractant protein-1. These changes were abolished by Mg but were only partially affected with ACEI. CsA promoted renal mRNA expression of fibrogenic molecules and extracellular matrices that were almost completely abolished by Mg but partially suppressed by ACEI. Similarly, CsA-induced chronic fibrotic lesion was markedly attenuated by Mg supplementation but was partially attenuated by ACEI. CONCLUSION: Mg supplementation abolished CsA-induced precedent interstitial inflammation possibly via inhibition of chemoattractants expression and consequently attenuated tubulointerstitial fibrosis. In this protective mechanism, factors independent of the renin-angiotensin system appears to be mainly involved.

Expression of nonmuscle myosin heavy chain B (SMemb) in rat allogeneic kidney transplantation.

BACKGROUND/AIM: Investigators have reported that the nonmuscle myosin heavy chain B (SMemb) expression is enhanced in various types of glomerular diseases which develop into nephrosclerosis. In renal transplantation, transplant glomerulitis is often recognized during acute rejection. Therefore, we hypothesized that SMemb plays important roles in acute kidney rejection. To evaluate the role of SMemb in the development of kidney rejection, we examined its expression in rat kidney transplantation models. METHODS: We used Lewis rats as recipients and Wistar rats as donors. Group I: controls; group II: isograft model; group III: allograft model; group IV: as group III +10 mg/kg/day of ciclosporin A (CsA), and group V: as group III + CsA administration for 5 days postoperatively. Histopathological and SMemb immunohistochemical studies were completed. RESULTS: Clear enhancement of SMemb expression was found on day 3 in group III. In groups I, II, IV, and V, SMemb was faintly expressed in the glomerular cells. However, after termination of CsA treatment, the SMemb expression increased. The expression of SMemb was higher in the allograft model than in either isograft or CsA-treated models. CONCLUSIONS: Immunohistological investigations show that the SMemb expression was significant from an early stage at which histopathological reactions were hardly identifiable. This, therefore, could be useful for an earlier diagnosis of acute rejection.

Expression of peroxisome proliferator-activated receptor (PPAR) in human prostate cancer.

BACKGROUND: Recent studies have demonstrated that peroxisome proliferator activator-receptors (PPAR)-gamma is expressed in some cancer cells such as breast, lung, and gastric cancer, and its ligand induces growth arrest of these cancer cells through apoptosis. However, the expression and localization of PPARs in prostate have not been examined. In this study, PPARs expression was investigated in human prostate cancer (PC), prostatic intraepithelial neoplasia (PIN), benign prostatic hyperplasia (BPH), and normal prostate (NP) tissues. METHODS: Tumor specimens were obtained from 156 patients with PC, 15 with PIN, 20 with BPH, and 12 patients with NP tissues. The expressions were investigated by RT-PCR and immunohistochemical methods. RESULTS: Immunoreactive PPAR-alpha and -beta were significantly apparent in PC tissues. Marked expressions of PPAR-alpha and -beta were also detected in PIN, BPH, and NP groups. However, very weak or no expression of immunoreactive PPAR-gamma was found in BPH and NP cases. In contrast, we found significant expression of immunoreactive PPAR-gamma in cancer cells in PC group and in PIN group. CONCLUSIONS: Our results demonstrated that PPAR-gamma is induced in PC, and suggest that PPAR-gamma ligands may mediate its own potent antiproliferative effect against PC cells through differentiation.

Antitumor Effect on Murine Renal Cell Carcinoma by Autologous Tumor Vaccines Genetically Modified with Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-6 Cells.

SUMMARY: The authors evaluted the efficacy of vaccination with murine renal cell carcinoma (Renca) secreting the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and interleukin-6 (IL-6) gene for the treatment of Renca tumor. Murine GM-CSF and murine IL-6 genes were introduced and expressed in Renca cells (Renca-GM-CSF and Renca-IL-6). For a prevaccination study, wild-type Renca cells were injected subcutaneously into Balb/c mice that had been vaccinated three times with inactivated wild-type Renca, Renca-GM-CSF, Renca-IL-6, or a mixture of Renca-GM-CSF and Renca-IL-6 cells 7, 14, and 21 days before this tumor inoculation. For vaccination experiments, Renca tumor-bearing (8 to 10 mm) mice were injected subcutaneously weekly for 3 weeks with inactivated wild-type Renca cells, or either one or a combination of Renca-GM-CSF and Renca-IL-6. A nonvaccinated control was included in all experiments. The animals were monitored for survival and tumor development for 8 weeks. Mice inoculated with wild-type Renca alone died from the tumor within 35 days. Renca-IL-6 grew slower than wild-type Renca (p < 0.05). No tumor was produced by Renca-GM-CSF. Prevaccination with the combination of Renca-GM-CSF and Renca-IL-6 prevented subsequently inoculated wild-type Renca from forming tumors, and prevaccination with either one of them, compared with prevaccination with wild-type Renca, retarded tumor growth and prolonged survival time. Tumor-bearing mice vaccinated with wild-type Renca died within 42 days. Vaccination with Renca-GM-CSF or Renca-IL-6 alone prolonged the survival time, but only Renca-GM-CSF drastically reduced the tumor size. Vaccination with the combination of them achieved complete remission. Neither of the cytokine-secreting cells enhanced the expression of MHC class I or II molecules. Autologous tumor cell vaccine secreting GM-CSF is effective in preventing and treating established tumors. Its efficacy is enhanced by the cosecretion of IL-6.