Effects of glucose-free dialysis solutions on human peritoneal mesothelial cells.

BACKGROUND: Glucose-free dialysis solutions may be more biocompatible owing to the physiological pH and the lack of glucose degradation products, and the effects can be reflected by the changes in some markers of peritoneal mesothelial cells (PMC). METHOD: Peritoneal effluents were collected in 17 CAPD patients after one daily exchange of glucose-containing dialysate to Nutrineal (1.1% amino acid-based PDF), and human PMC were cultured from peritoneal effluent and treated with various peritoneal dialysis (PD) solutions. RESULTS: The level of cancer antigen 125 (CA125) in peritoneal effluent increased significantly after using Nutrineal for 3 months (p = 0.045), whereas that of procollagen I peptide (PICP) remained unaltered. Production of CA125 by human PMC showed a time-responsive increase after stimulation with Nutrineal and Extraneal (icodextran-based PDF). An increased expression of CA125 was observed in cultured human PMC treated with various PD solutions, and the increase induced by Nutrineal was lower than that induced by 4.25% Dianeal and Extraneal. A lower increase was also observed for lactate dehydrogenase (LDH). The levels of heat shock protein 70 (HSP70), however, were not altered. CONCLUSION: Nutrineal is more biocompatible to the peritoneal membrane than the conventional PD solutions tested herein, reflected by both the in vivo and in vitro response of CA125.

Correlation of plasma homocysteine level with arterial stiffness and pulse pressure in hemodialysis patients.

Elevated plasma homocysteine, arterial stiffness, and increased pulse pressure (PP) are independently associated with higher cardiovascular risk in patients with end-stage renal disease. The aim of this study is to investigate the influence of plasma homocysteine on arterial stiffness and PP in hemodialysis (HD) patients. One hundred and nine HD patients were stratified into three groups by plasma homocysteine levels: low (11.2-20.8 micromol/L), middle (21.2-25.1 micromol/L), and high tertiles of plasma homocysteine (Hcy) group (25.2-43.9 micromol/L). Using a computerized oscillometry, we measured the arterial stiffness index (ASI) and blood pressure (BP) hemodynamic parameters in the brachial artery. The high Hcy group exhibited a higher ASI (110.4+/-129.5 versus 46.2+/-17.5, mean+/-S.E., P<0.01), PP (59.7+/-23.1 versus 43.3+/-16.3 mmHg, P<0.01), and age (57.8+/-14.1 versus 49.9+/-12.7 years, P<0.05) compared with the low Hcy group. Plasma homocysteine was significantly correlated with ASI (r=0.25, P<0.001), PP (r=0.33, P<0.001), systolic BP (r=0.31, P<0.001), and age (r=0.24, P<0.05). Serum ferritin was significantly correlated with ASI (r=0.24, P<0.05) and PP (r=0.23, P<0.05). ASI was also correlated with PP (r=0.64, P<0.001). Multiple regression analyses showed that both plasma homocysteine and serum ferritin had significant associations with ASI (beta=4.246, P=0.007 and beta=0.024, P=0.006, respectively), and with PP (beta=1.089, P=0.002 and beta=0.005, P=0.005, respectively) independent of other classic risk factors for atherosclerosis. In conclusion, plasma homocysteine, along with serum ferritin, may act as an important predictor for arterial stiffness and PP in HD patients.

Role of lipid control in diabetic nephropathy.

Patients with diabetic nephropathy are known to be associated with many lipoprotein abnormalities, including higher plasma levels of very low-density lipoprotein, low-density lipoprotein and triglycerides, and lower levels of high-density lipoprotein. Many studies have reported that lipids may induce both glomerular and tubulointerstitial injury through mediators such as cytokines, reactive oxygen species, chemokines, and through hemodynamic changes. Clinical studies in patients with diabetic nephropathy showed that lipid control can be associated with an additional effect of reduction in proteinuria. Experimental studies demonstrated that lipid-lowering agents exerted a certain degree of renoprotection, through both indirect effects from lipid lowering and a direct effect on cell protection. Therefore, lipid control appears to be important in the prevention and treatment of diabetic nephropathy. Diabetic nephropathy has become the leading cause of end-stage renal failure in many countries, including Taiwan. One of the major risk factors for the development and progression of diabetic nephropathy is dyslipidemia. In this paper we will review the role of lipid in mediating renal injury and the beneficial effects of lipid control in diabetic nephropathy.

San-Huang-Xie-Xin-Tang reduces lipopolysaccharides-induced hypotension and inflammatory mediators.

San-Huang-Xie-Xin-Tang (SHXT) is a traditional Chinese medicinal formula containing Coptidis rhizoma, Scutellariae radix and Rhei rhizoma. The present study aimed to determine the preventive effects of standardized SHXT on lipopolysaccharides (LPS)-induced arterial hypotension, protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), cytokines formation and prostaglandin E2 (PGE2) production. LPS-induced activation of iNOS has been recognized to increase cytokines and nitric oxide, some of them play predominant roles in sepsis. Intravenous injection of LPS (10 mg/kg) caused a marked decrease of the mean arterial pressure in normotensive rats. However, the LPS-induced arterial hypotension was inhibited by SHXT (0.01 and 0.03 g/kg), when it was given 30 min before LPS. Moreover, plasma level of cytokines and PGE2 were lowered by SHXT. In RAW 264.7 cells, SHXT (20-200 microg/ml) dose-dependently inhibited LPS (1 microg/ml)-induced iNOS and COX-2 expression, and it also significantly decreased LPS-induced cytokines in a dose-dependent manner. In conclusion, our data suggest that SHXT prevented LPS-induced arterial hypotension, which might be mediated through its inhibition activities on the expression of iNOS and COX-2, cytokines formation and PGE2 production. Therefore, its protection activity against LPS-induced arterial hypotension and inflammatory mediators release might be beneficial in the treatment of endotoxin shock and/or associated inflammation.

HCV infection complicated with nephrotic syndrome, immune complex crescentic glomerulonephritis and acute renal failure: a case report.

There is ample evidence suggesting that hepatitis C virus (HCV)-associated autoimmunity plays a role in a broad spectrum of autoimmune diseases, which are usually overlooked. We report on a case of nephrotic syndrome, palpable purpura, cryoglobulinemia, hypocomplementemia, and acute renal failure complicated by immune complex glomerulonephritis (GN). The patient is a 64-year-old man with HCV infection, who was initially considered to present only an HCV-associated cryoglobulinemic GN. However, renal biopsy revealed a "full house" immune complex crescentic GN, which led to our subsequent investigation. The attending clinicians faced what is a common dilemma, where an HCV-associated autoimmune disease inevitably switches to a lupus-like GN. Hence, we also discuss treatment.

Pravastatin suppress superoxide and fibronectin production of glomerular mesangial cells induced by oxidized-LDL and high glucose.

Pravastatin is a potent inhibitor of HMG-CoA reductase and is effective in lowering serum lipid levels. Recent studies have shown that pravastatin also reduces oxidative modification of LDL and decreases albuminuria in patients with diabetes. To determine the possible benefit of pravastatin on the diabetic kidney, we have measured the effects of pravastatin on the proliferation and the production of superoxide and fibronectin, and the expression of fibronectin mRNA of glomerular mesangial cells stimulated by oxidized-LDL and high glucose. Our results demonstrated that the [(3)H]-labeled thymidine uptake of mesangial cells decreased after oxidized-LDL stimulation (50 microg/ml, 6 h) and increased after high glucose stimulation (25 mM, 48 h). The production of superoxide and fibronectin and the expression of fibronectin mRNA of glomerular mesangial cells were all significantly increased after stimulation with either oxidized-LDL or high glucose, or the combination of oxidized-LDL and high glucose. Pravastatin (100 microM, 48 h) alone had no effect on unstimulated cells. However, pravastatin significantly reversed thymidine uptake, inhibited the production of superoxide and fibronectin, and inhibited the expression of fibronectin mRNA of glomerular mesangial cells after stimulation with either oxidized-LDL or high glucose. Our results indicate that pravastatin may effect as an antioxidant and may suppress fibronectin synthesis of glomerular mesangial cells in diabetic patients with hyperlipidemia.

Risk factors and risk for mortality of mild hypoparathyroidism in hemodialysis patients.

Relative hypoparathyroidism (parathyroid hormone [PTH] < or = 200 pg/mL) is prevalent in hemodialysis (HD) patients, with unknown pathogenesis and prognosis. Thus, to clarify risk factors and prognosis of time-dependent relative hypoparathyroidism in HD patients, a retrospective cohort study was performed for 126 HD patients with four or more PTH determinations and no previous total or subtotal parathyroidectomy. Values for intact PTH, ionized calcium, phosphate, magnesium, albumin, creatinine, urea reduction ratio (URR), glucose, hemoglobin A1c (HbA1c), aluminum, and 1,25(OH)2D were obtained at enrollment and at some time during follow-up. The prevalence of relative hypoparathyroidism at entry was 76 of 126 patients (60.3%). Univariate analysis showed that patients with hypoparathyroidism were older, more likely to have diabetes, and had greater ionized calcium levels and lower phosphate, albumin, blood urea nitrogen (BUN), and creatinine levels. Patients with diabetes were older and had a shorter duration of dialysis therapy and lower PTH, phosphate, albumin, BUN, and creatinine levels and URRs. Conversely, multivariate analysis showed that PTH levels at entry were associated directly with creatinine levels and inversely with age and ionized calcium levels (but not diabetes). During follow-up, PTH levels fluctuated concomitantly with ionized calcium and phosphate levels over time in all patients. Time-dependent PTH levels were associated directly with duration of dialysis therapy and use of vitamin D and phosphate and albumin levels, but inversely with age and ionized calcium and magnesium levels (but not glucose or HbA1c levels). Interestingly, time-dependent PTH levels were independently associated with survival after adjusting for traditional risk factors (diabetes, age, albumin and creatinine levels, and URR) and duration of dialysis therapy. We conclude that in HD patients, relative hypoparathyroidism was not associated with diabetes per se. Time-dependent PTH levels were associated with age, duration of dialysis, and levels of ionized calcium, phosphate, albumin, and magnesium. Moreover, relative hypoparathyroidism at entry and lower time-dependent PTH levels predict mortality.

Association of death from renal failure with calcium levels in drinking water.

The possible association between the risk of death from renal failure (RF) and the levels of calcium in drinking water from municipal supplies was investigated in a matched case-control study in Taiwan. Characteristics for all eligible RF-related deaths (2469 cases) among Taiwan residents from 1991 through 2000 were compared with those of people who died from other causes (2469 controls). The levels of calcium in the drinking water of these residents were determined from data obtained from the Taiwan Water Supply Corporation (TWSC). The controls were pair-matched to the RF-related cases by year of birth and death. The adjusted odds ratios (ORs) (95% confidence interval, CI) for RF-related deaths were 1.21 (1.03-1.43) for the group with water calcium levels between 25.1 and 43.0 mg/L and 1.34 (1.12-1.60) for the group with calcium levels of 43.3 mg/L or more. There was a significant trend for increased risk of death from RF with higher calcium levels in drinking water.

Anti-cancer effect of liriodenine on human lung cancer cells.

Our previous work demonstrated that liriodenine, an isoquinoline alkaloid isolated from plant species of many genera, exhibits a potent cytotoxic effect on various types of human cancer cells. In this study, we investigated the effect of liriodenine on the growth and viability of human lung cancer cells and addressed the underlying mechanism of action. We found that liriodenine suppressed proliferation of A549 human lung adenocarcinoma cells in a dose- and time-dependent manner. Flow cytometric analysis showed that liriodenine blocked cell cycle progression at the G2/M phase. Induction of G2/M arrest by liriodenine was accompanied by reduction of G1 cyclin (D1) and accumulation of G2 cyclin (B1). In vitro kinase activity assay demonstrated that the enzymatic activity of the cyclin B1/cyclin-dependent kinase 1 complex was reduced in liriodenine-treated cells. More importantly, incubation with liriodenine led to activation of caspases and apoptosis in A549 cells. The apoptosis-inducing activity of liriodenine was more apparent when cells were cultured under serum-free conditions. Our results indicate that liriodenine exerts potent anti-proliferative and apoptosis-inducing effects on human lung cancer cells.

Changes of renal cortical Na-K ATPase activity, protein, and mRNA expression in ureteral obstruction.

Decreased renal Na-K ATPase activity in ureteral obstruction contributes to tubular sodium reabsorption defect in obstructive nephropathy. The integrated changes on the enzyme activity, protein number, and mRNA level of renal cortical Na-K ATPase, in response to bilateral or unilateral ureteral obstruction (BUO & UUO), were studied in rabbits. Ouabain-sensitive Na-K ATPase activities of renal cortex were significantly decreased at 24 h after BUO and further decreased at 48 h. The unobstructed contra-lateral kidney had significantly higher Na-K ATPase activity compared to the obstructed side. Immunoblots of Na-K ATPase alpha and beta subunits protein were both decreased at 24 h of BUO and further decreased at 48 h. The levels of Na-K ATPase beta subunit mRNA showed to be significantly decreased at 12 h after obstruction and further decreased at 24 and 48 h. However, the levels of alpha subunit mRNA were not changed as that of beta subunit throughout the study period. This study also used a newly developed method for release of obstruction. All the parameters studied above recovered to variable extents after release of the ureteral obstruction. In summary, decreases in Na-K ATPase activity, protein, and mRNA in obstructed kidney are specific cellular responses to ureteral obstruction. The degree of down-regulation is related to the duration of obstruction. The reduced activity of Na-K ATPase can be explained by decreased enzyme protein. However, the discordant findings in Na-K ATPase subunits protein amounts and mRNA changes suggest that renal Na-K ATPase subunits have different cellular regulatory processes to obstruction, in which transcriptional, translational and even intra-cytoplasmic processing may be involved.

Signaling and regulatory mechanisms of integrinalpha3beta1 on the apoptosis of cultured rat podocytes.

Integrin is the major adhesion molecule for the attachment of podocytes to the glomerular basement membrane, and integrins have been shown to play a major role in the regulation of cell survival. In this study, the authors investigated the apoptosis and its related signal pathways to integrin in cultured rat podocytes. Apoptosis was detected with the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique. Cytochrome c was examined by immunohistochemical stain, and Fas, Fas ligand, Bax, Bcl-2, and ERK activation (p-ERK/ERK) were analyzed by Western blotting analysis. The results demonstrated that the integrin antagonist, Gly-Arg-Gly-Asp (GRGD), increased the percentage of cells with apoptosis (from 0.9+/-0.5% to 27.2+/-9.9%, P < 0.01). Inhibition of protein tyrosine kinase with genistein also caused apoptosis of podocytes (from 0.9+/-0.5% to 26.0+/-8.7%, P < 0.01). In GRGD-treated cells, cytochrome c was found released into cytoplasm by immunohistochemical study and the Bax expression was upregulated, whereas Bcl-2 expression was not changed. Fas was not expressed in both control and GRGD-treated podocytes, although Fas ligand was upregulated in GRGD-treated cells. ERK activation was also found to be increased in GRGD-treated cells. The results indicated that alpha3beta1integrin is necessary for the prevention of the apoptosis of cultured rat podocytes, and that the signaling involves the Bax, Bcl-2, and cytochrome c pathways.

Reduced podocyte expression of alpha3beta1 integrins and podocyte depletion in patients with primary focal segmental glomerulosclerosis and chronic PAN-treated rats.

Integrins attach cells to extracellular matrix (ECM) and mediate signals from ECM to cells or from cells to ECM. They regulate cell functions, including adhesion, migration, cell cycle regulation, and differentiation. Podocytes may detach from the glomerular basement membrane (GBM) and be excreted in the urine, and proteinuria is found in patients with primary focal segmental glomerulosclerosis (FSGS); both may be associated with loss of alpha3beta1integrins. In this study, we have examined the podocyte number in patients with primary FSGS and normal controls, and the alpha3- and beta1-integrin subunits expression of podocytes in patients with primary FSGS and chronic puromycin aminonucleoside (PAN)-treated rats by the morphometric, immunoperoxidase histochemical, and immunoelectron microscopic examination. We also measured their expression serially in rats that received repeated PAN injection. The results showed that the podocyte number was significantly decreased in patients with primary FSGS than in normal control (P < 0.05). The immunostaining score showed that both alpha3- and beta1-integrin subunits on podocytes in patients with primary FSGS were significantly lower than in normal controls (both P < 0.01). The number of immuno-gold particles of alpha3- and beta1-integrins at the effaced foot process area of patients with primary FSGS were also significantly decreased than that of normal controls (both P < 0.05). The immunostaining score of both alpha3- and beta1-integrin subunits was negatively correlated with the degree of glomerular sclerosing score and the amount of daily protein loss, and they were positively correlated with the number of podocytes. Chronic 12-week PAN-treated rats showed similar findings with decreased immunostaining expression and immuno-gold particles of alpha3-integrin on podocytes than in normal control (both P < 0.05). The chronic PAN-treated rats also showed a trend toward gradually decreased immunostaining expression of alpha3-integrin subunit on podocyte during the progress from normal to FSGS state. These studies indicate that podocyte expression of alpha3- and beta1-integrin subunits is significantly reduced in humans with primary FSGS and chronic PAN-treated rats, before the morphological changes of FSGS are observed. The decreased podocyte expression of alpha3beta1 integrins is closely related with podocyte depletion, glomerular sclerosis, and daily protein loss in patients with primary FSGS.

Decreased synthesis of glomerular adrenomedullin in patients with IgA nephropathy.

Adrenomedullin (AM) immunostaining and gene expression have seldom been measured in human kidneys. Because previous studies have shown that AM exerts antiproliferative effects on rat mesangial cells in vitro and that urine AM levels are decreased in patients with chronic glomerulonephritis, we measured glomerular AM and its gene expression in patients with primary IgA nephropathy (IgAN). Glomerular AM was measured by immunohistochemical staining, and glomerular AM mRNA was measured by in situ hybridization. Plasma and urine AM were measured by radioimmunoassay. The results showed that both the intensity of immunostaining for glomerular AM and the glomerular expression of AM mRNA were significantly decreased in IgAN patients compared with normal controls (both P < .05). Similar results were not observed in patients with non-IgA MsPGN. Glomerular AM immunostaining and glomerular AM mRNA expression were significantly correlated ( P < .001), and both were negatively correlated with the number of glomerular cells ( P < .05 and < .01, respectively). Both glomerular AM immunostaining and glomerular AM mRNA expression were correlated with urine AM levels (both P < .001), but not with plasma AM levels. The urine AM level was significantly lower in IgAN patients than in normal controls ( P < .01), whereas the plasma level was not different between the 2 groups. Our findings indicate that glomerular production of AM was decreased in patients with IgA nephropathy and that this lack of glomerular AM may be related to the pathogenesis of this mesangial disease.

Alterations of glomerular and extracellular levels of glutathione peroxidase in patients and experimental rats with diabetic nephropathy.

To investigate the status and role of glutathione peroxidase (GPX) in diabetic nephropathy, we measured GPX in the plasma and urine of 14 patients with diabetic glomerulosclerosis (DGS) and measured glomerular GPX immunostaining in these patients and in rats with streptozotocin-induced diabetes of varying duration. Plasma GPX levels were significantly lower in DGS patients than in diabetic patients without nephropathy (P < .05) or normal controls (P < .01). Urinary GPX concentrations were also significantly lower in DGS patients than in diabetic patients without nephropathy or normal controls (both P < .05). Immunostaining of glomerular GPX was significantly less in DGS patients than in normal controls (P < .05) and was negatively correlated with the glomerular sclerosis score and the index of mesangial expansion. Serial examination of glomerular GPX in diabetic rats showed that immunostaining scores for glomerular GPX in rats were significantly lower than those in normal control rats after 1 and 3 months' duration of diabetes, and staining scores were also significantly lower in rats killed after 3 months of diabetes than in those killed after 1 week. In conclusion, our study demonstrates that GPX concentrations in plasma, urine, and glomeruli are decreased in individuals with DGS and that the immunostaining of glomerular GPX decreases progressively.

Effects of pravastatin on superoxide and fibronectin production of mesangial cells induced by low-density lipoprotein.

Pravastatin is a potent inhibitor of HMG-CoA reductase and is effective in lowering serum lipid levels. Recent studies have shown that pravastatin also reduces the oxidative modification of low-density lipoprotein (LDL). To determine whether pravastatin has a direct effect on glomerular mesangial cells, we have measured the effects of pravastatin on the production of superoxide and fibronectin of glomerular mesangial cells stimulated by LDL. Our results demonstrated that the superoxide production of mesangial cells increased after LDL stimulation (100 microg/ml for 4 h) and that the superoxide production was significantly suppressed by either superoxide dismutase (SOD; 500 U/ml for 36 h; p < 0.01) or pravastatin (80 microM for 36 h; p < 0.05). The production of fibronectin was also increased after LDL stimulation which was also significantly suppressed by either SOD (p < 0.01) or pravastatin (p < 0.01). SOD or pravastatin alone had no effect on the unstimulated cells. Our results indicate that pravastatin may have a direct effect as an antioxidant and suppresses the fibronectin synthesis of glomerular mesangial cells independent of its hypolipidemic effect.

Does amino acid-based peritoneal dialysate change homocysteine metabolism in continuous ambulatory peritoneal dialysis patients?

OBJECTIVE: We examined whether amino acid-based peritoneal dialysate that contains 85 mg/dL L-methionine affects homocysteine (Hcy) metabolism. DESIGN: The study enrolled 17 adult CAPD patients (11 men, 6 women) who had been receiving CAPD for at least 6 months and who had low serum albumin levels (<3.7 g/dL). Diet was not specifically changed. All of the study patients received daily 4-exchange CAPD treatment, and they used Nutrineal (Baxter Healthcare, Deerfield, IL, U.S.A.) as one of their daily exchanges (first or second exchange). Blood samples were collected every 2 weeks, and Hcy was measured. RESULTS: After use of Nutrineal, serum albumin was unchanged, but blood urea nitrogen (BUN) and total protein were increased. Before the study began, 1 patient had a very high Hcy level (256 micromol/L); his data were excluded from the analysis. In the remaining 16 patients, baseline Hcy was 24.4 +/- 7.0 micromol/L. Levels of Hcy progressively increased with the use of Nutrineal: 28.1 +/- 6.2 micromol/L in week 2, 28.4 +/- 7.1 micromol/L in week 4, 29.1 +/- 7.6 micromol/L in week 6, 29.3 +/- 9.0 micromol/L in week 8, 27.5+/-9.7 micromol/L in week 10, and 30.3 +/- 8.2 micromol/L in week 12. CONCLUSIONS: Nutrineal might help to replenish daily protein loss, but it also increased formation of Hcy and, therefore, the potential risk of cardiovascular illness. Further studies will be needed to examine the effect of folic acid and vitamin B(12) supplementation in the rescue of that Hcy increase, and also a possible correlation with methylenetetrahydrofolate reductase gene polymorphism.

Beta-hydroxybutyrate-induced growth inhibition and collagen production in HK-2 cells are dependent on TGF-beta and Smad3.

BACKGROUND: Ketonuria is common in diabetes. The major form of ketone body is beta-hydroxybutyrate (beta-HB), which is metabolized by the proximal tubule. Transforming growth factor beta (TGF-beta) and tubulopathy are important in diabetic nephropathy. Thus, the role of TGF-beta and the downstream Smad3 in beta-HB-induced effects in the human proximal tubule (HK-2 cell) was studied. METHODS: Effects of beta-HB (0.1 to 10 mmol/L) on HK-2 cells were determined for: proliferation, cell cycle distribution, collagen production, tubular transdifferentiation [expression of alpha-smooth muscle actin (alpha-SMA) protein], TGF-beta, Smad2/3, p21WAF1, and p27kip1. RESULTS: Beta-HB (0.1 to 10 mmol/L) dose dependently decreased proliferation, arrested the cells in G0/G1 phase of the cell cycle, and increased p21WAF1/p27kip1 protein expression at 48 hours (without affecting p21WAF1/p27kip1 mRNA and transcription). beta-HB (1 mmol/L) increased p21WAF1/p27kip1 protein half-lives. Beta-HB (1 mmol/L) increased TGF-beta transcription at 24 hours and TGF-beta1 mRNA/bioactivity at 48 hours. Beta-HB (1 mmol/L) increased nuclear Smad2/3 protein expression and increased collagen production (without affecting tubular transdifferentiation), which were reversed by Smad7, dominant-negative Smad3, and N-acetylcysteine. Dominant-negative Smad3 reversed beta-HB-induced TGF-beta transcription at 24 hours, and reversed TGF-beta1 bioactivity at 48 hours. Dominant-negative Smad3 reversed beta-HB-induced p21WAF1/p27kip1 protein expression at 48 hours. Finally, N-acetylcysteine, TGF-beta antibody, Smad7, and dominant-negative Smad3 reversed beta-HB (1 mmol/L)-induced growth inhibition at 48 hours. CONCLUSION: Beta-HB activated Smad 2/3 by oxidative stress. TGF-beta and Smad3 mediate beta-HB-induced cell cycle-dependent growth inhibition while Smad3 mediate beta-HB-induced collagen production and p21WAF1/p27kip1 protein expression in HK-2 cells. Moreover, beta-HB increased p21WAF1/p27kip1 protein expression by increasing p21WAF1/p27kip1 protein stability.

1-alpha,25-Dihydroxyvitamin D3 regulates inducible nitric oxide synthase messenger RNA expression and nitric oxide release in macrophage-like RAW 264.7 cells.

The expression of inducible nitric oxide synthase (iNOS) expression and release of nitric oxide (NO) from macrophages are markedly increased in granulomatous infections. Activation of macrophages 1alpha-hydroxylase results in an increase of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, the significance of this increased production is not completely understood. In this study, we analyzed 1,25(OH)(2)D(3) and NO production in patients with tuberculosis infection and hypercalcemia and used lipopolysaccharide (LPS) to stimulate RAW 264.7 cells in an attempt to assess iNOS expression and gaseous NO production regulated by 1,25(OH)(2)D(3). Peroxynitrite (OONO(-)) production and lactate dehydrogenase activity were also examined. Without additional stimulation, peripheral-blood mononuclear cells (PBMCs) from patients with tuberculosis converted more 25-hydroxyvitamin D(3) to 1,25(OH)(2)D(3) than did those from normal controls. These PBMCs released less NO than did those from control subjects, at baseline and in the stimulated state. We found that 1,25(OH)(2)D(3) dose-dependently inhibited iNOS messenger RNA expression of the LPS-stimulated RAW 264.7 cells and also significantly reduced the gaseous NO release and OONO(-) production. Paralleling the 1,25(OH)(2)D(3)-induced inhibition of NO release were reductions in OONO(-) and LDH production. In conclusion, 1,25(OH)(2)D(3) inhibited iNOS expression and reduced NO production by LPS-stimulated macrophages in the range of physiological doses. Inhibition of the NO surge was coupled with a reduction in OONO(-) and LDH production. Increased 1,25(OH)(2)D(3) production and decreased release of NO from the PBMCs of patients with tuberculosis and hypercalcemia were also noted. We propose that 1,25(OH)(2)D(3) production by macrophages may protect themselves against oxidative injuries caused by the NO burst. In the case of tuberculosis infection, increased 1,25(OH)(2)D(3) synthesis may further contribute to the development of an unwanted phenomenon-hypercalcemia.

Leptin and connective tissue growth factor in advanced glycation end-product-induced effects in NRK-49F cells.

Previously, we showed that Janus kinase 2 (JAK2) is important in advanced glycation end-product (AGE)-induced effects in renal interstitial (NRK-49F) fibroblasts. Leptin is a JAK2-activating cytokine via the long form leptin receptor (Ob-Rb). Leptin and connective tissue growth factor (CTGF) may be involved in renal fibrosis. However, the relationship between leptin and CTGF in terms of AGE-induced effects remains unknown. Thus, the effects of AGE (150 microg/ml) and leptin on mitogenesis, CTGF and collagen expression in NRK-49F cells were determined. We found that leptin and AGE increased mitogenesis and type I collagen protein expression at 3 and 7 days, respectively. AGE increased leptin mRNA and protein expression at 2-3 days. AGE increased CTGF mRNA and protein expression at 3-5 days. AG-490 (JAK2 inhibitor) abrogated AGE-induced leptin mRNA and protein expression at 2-3 days. AG-490 and Ob-Rb anti-sense oligodeoxynucleotides (ODN) abrogated AGE-induced CTGF mRNA and protein expression at 3-5 days. AG-490 and CTGF anti-sense ODN abrogated AGE-induced mitogenesis and collagen protein expression at 7 days. Additionally, leptin dose (0.2-1 microg/ml) and time (1-2 days)-dependently increased CTGF protein expression. AG-490 abrogated leptin (1 microg/ml)-induced CTGF protein expression at 2 days. AG-490 and CTGF anti-sense ODN abrogated leptin-induced mitogenesis and collagen protein expression at 3 days. We concluded that AGE induced JAK2 to increase leptin while leptin induced JAK2 to increase CTGF-induced mitogenesis and type I collagen protein expression in NRK-49F cells. Additionally, AGE-induced mitogenesis and type I collagen protein expression were dependent on leptin-induced CTGF.