Role of D2 dopamine receptor in adrenal cortical cell proliferation and aldosterone-producing adenoma tumorigenesis.

Aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia are the two characteristic types of primary aldosteronism. Dysregulation of adrenal cortical cell proliferation contributes to both diseases. We previously demonstrated that APA expressed less dopamine D2 receptor than the respective non-tumor tissue and might contribute to the overproduction of aldosterone. As activation of D2 receptor inhibits the proliferation of various cells, downregulation of D2 receptor in APA may play a role in the tumorigenesis of APA. In this study, we demonstrate that D2 receptor plays a role in angiotensin II (AII)-stimulated adrenal cortical cell proliferation. The D2 receptor agonist, bromocriptine, inhibited AII-stimulated cell proliferation in primary cultures of the normal human adrenal cortex and APA through attenuating AII-induced phosphorylation of PK-stimulated cyclin D1 protein expression and cell proliferation. D2 receptor also inhibited AII-induced ERK1/2 phosphorylation. Our results demonstrate that, in addition to inhibiting aldosterone synthesis/production, D2 receptor exerts an anti-proliferative effect in adrenal cortical and APA cells by attenuating PKCµ and ERK phosphorylation. The lower level of expression of D2 receptor in APA may augment cell proliferation and plays a crucial role in the tumorigenesis of APA. Our novel finding suggests a new therapeutic target for primary aldosteronism.

In rat renal fibroblasts, mycophenolic acid inhibits proliferation and production of the chemokine CCL2, stimulated by tumour necrosis factor-alpha.

BACKGROUND AND PURPOSE: Renal fibroblasts play a pivotal role in the development of tubulointerstitial fibrosis, a condition highly predictive of progression towards end-stage renal disease. The present study investigated the anti-mitogenic and anti-inflammatory effects of an inhibitor of inosine monophosphate dehydrogenase, mycophenolic acid (MPA) and the mechanisms underlying its action in normal rat kidney fibroblasts (49F cells). EXPERIMENTAL APPROACH: Proliferation of 49F cells was studied by tetrazole 3-(4, 5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT) test, bromodeoxyuridine incorporation and flow cytometry. The cyclins, tumour suppressor genes and phospho-mitogen-activated protein kinases (MAPKs) were semiquantified by immunoblotting. Apoptosis was measured by quantifying the fragmented DNA and the activity of caspase 3. The monocyte chemokine CCL2 was measured by ELISA. The mRNA expression of CCL2 was measured by real-time PCR. KEY RESULTS: Mycophenolic acid dose-dependently inhibited steady-state proliferation of 49F cells by up-regulation of p21, p27 and p53, in association with a decrease in cyclins D2 and E. Treatment with MPA also triggered apoptosis of 49F cells by activating the caspase 3 cascade. Furthermore, MPA attenuated tumour necrosis factor-alpha-induced CCL2 expression through down-regulation of p38 MAPK, but not that of ERK1/2 or JNK. CONCLUSIONS AND IMPLICATIONS: The anti-mitogenic and anti-inflammatory effects of MPA were mediated by up-regulation of cell cycle inhibitors and pro-apoptotic signals, and by suppression of p38 MAPK pathway respectively. This dual effect of MPA may form the rationale for animal or clinical trials for the treatment of fibrotic renal diseases.

Evidence-based consensus development and reduction of rate of endoscopic thoracic sympathectomy: a national study.

BACKGROUND: Endoscopic thoracic sympathectomy (ETS) was overused for treating patients with hyperhidrosis in Taiwan. OBJECTIVES: To determine whether use of evidence and a consensus-building exercise can reduce rates of surgery. METHODS: We invited all surgeons in Taiwan who performed five or more ETSs per year for treating patients with hyperhidrosis to join this study. A structured questionnaire was mailed to surgeons asking about their management decisions based on clinical scenarios. Then we provided surgeons with evidence synopses and used the Delphi method to reach consensus. We analyzed healthcare utilization data during 2000 to 2005 and calculated total numbers of ETSs performed per 12 months to examine the effect on surgeons' behavior. RESULTS: Of 155 surgeons invited, 61 (40%) completed this study. They agreed that observation or topical therapy was appropriate for patients with mild palmar hyperhidrosis, whereas ETS was appropriate for children, adolescents, and young and middle-aged adults who had severe symptoms. Surgeons became more willing to recommend botulinum toxin injection after we provided evidence synopses. We found a 52% reduction in mean total ETSs per 12 months in surgeons receiving evidence synopses. A higher percentage of reduction occurred in patients younger than 12 and aged 60 and older. CONCLUSION: Evidence-based consensus development is helpful in decreasing overuse of ETS in treating patients with hyperhidrosis in Taiwan.

A programme of accelerated medical education in Taiwan.

BACKGROUND: Graduates of the 7-year undergraduate medical curriculum in Taiwan are often deficient in clinical skills. AIMS: To implement and assess a programme of accelerated clinical education. METHOD: The Department of Primary Care Medicine at the National Taiwan University College of Medicine implemented a programme shortening the undergraduate clinical curriculum from 3 to 2 years and giving students more clinical responsibility. Students were prepared for clinical rotations with a 1-month clinical skills course. Core clinical rotations were redesigned to be more participatory. The programme included 1 year of a postgraduate, rotating residency. Self-selected students with adequate grades, recommendations and performance on an interview participated in the programme. None of them dropped out. RESULTS: Compared with their traditionally instructed cohorts, graduates of the accelerated programme ( approximately 10% of each class) were more likely to pass national boards (100% versus 80-97%) and were rated as more proficient on 9 of the 10 different clinical performance parameters (p < 0.01 by sign test). Sixty-nine percent reported being satisfied or very satisfied with the programme. CONCLUSION: A pilot programme of accelerated medical education at National Taiwan University that included clinical skills instruction, mentor-style classes and active learning techniques resulted in satisfactory outcomes for the students selected for the programme.

D4 dopamine receptor enhances angiotensin II-stimulated aldosterone secretion through PKC-epsilon and calcium signaling.

Aldosterone secretion is subjected to dopaminergic regulation. Our previous study showed that both human D2 and D4 dopamine receptors (D2R and D4R) modulate aldosterone secretion, but in opposing directions. The inhibitory effect of D2R is mediated by attenuating protein kinase C-micro (PKC-micro) and calcium-dependent signaling. The mechanism of D4R effect on angiotensin II (AII)-stimulated aldosterone secretion is explored in this study. Experiments were done with primary human adrenal cortical cells and human adrenocarcinoma (NCI-H295R) cells. Activation of different PKC isoforms was detected by specific phospho-PKC antibodies and PKC translocation. The role of calcium-dependent signaling was examined by measuring the cytoplasmic inositol 1,4,5-triphosphate (IP(3)) and calcium ([Ca(2+)](i)). The D4R agonist PD-168,077 enhanced AII-stimulated aldosterone synthesis and secretion as early as 30 min following exposure independently of the modulation of aldosterone synthase (CYP11B2) transcription. CYP11B2 mRNA level elevated by AII was augmented by D4R in the later period. These effects were reversed by the D4R antagonist L-745,870. AII activated PKC-alpha/betaII, -epsilon, and -micro but not PKC-delta, -theta, or -zeta/lambda of H295R cells. The D4R agonist selectively enhanced AII-stimulated PKC-epsilon phosphorylation and its translocation to the cell membrane. Furthermore, the D4R agonist enhanced the AII-stimulated elevation of intracellular IP(3) and [Ca(2+)](i). Inhibition of PKC-epsilon translocation by the PKC-epsilon-specific inhibitory peptide attenuated AII-stimulated aldosterone secretion, CYP11B2 mRNA expression, and elevation of intracellular IP(3) and [Ca(2+)](i). We conclude that D4R augmented aldosterone synthesis/secretion induced by AII. The mechanisms responsible for this augmentation are mediated through enhancing PKC-epsilon phosphorylation and [Ca(2+)](i) elevation.

The learning of 7th year medical students at internal medical--evaluation by logbooks.

INTRODUCTION: The purpose of this study was to understand the learning of internal medicine of 7th year medical students through records of the "Learning Passport". MATERIALS AND METHODS: Between June 2005 and June 2006, data from the learning passport (a type of logbook) of 207 7th year medical students at the Department of Internal Medicine, National Taiwan University Hospital (NTUH) were collected. RESULTS: Among the 19 symptoms/signs listed in the logbook, a large number of students did not learn well on low back pain, skin rash, oedema, oliguria and anxiety/depression; only a few students rated themselves as knowledgeable about anxiety/depression, malaise, skin rash, headache and anorexia. Among the 16 diseases listed, a large number of students did not learn well on chronic obstructive pulmonary disease, stroke, hypertension, coronary artery disease and cirrhosis; only a few students rated themselves as knowledgeable about shock, respiratory failure, consciousness disturbance, sepsis and renal failure. Among the 21 physical examination skills listed, a large number of students did not learn well on the male genitalia, eyes, cognitive status, mental state and the digital rectal examination; only a few students rated themselves as fully competent about cognitive status, mental state, eyes, neurology examination and ENT examination. Among the 11 laboratory skills and image interpretation skills listed, a large number of students did not learn well on blood smear, Gram's stain and specimen sampling; only a few students rated themselves as fully competent about the interpretation of brain computed tomographic (CT) scan, blood smear and Gram's stain. Among the 12 procedures and therapeutic skills listed, a large number of students did not learn well on observation of lumbar puncture, basic cardiopulmonary resuscitation (CPR) and aseptic procedure; only a few students rated themselves as fully competent about basic CPR and transfusion management. CONCLUSIONS: The weak points of intern training conducted by the Department of Internal Medicine, NTUH were revealed by analysis obtained from their logbooks. Thus, we need to strengthen the learning of the interns in these specific parts and assess their performance based on the use of portfolios.

Endothelin-1 activates MAPKs and modulates cell cycle proteins in OKP cells.

BACKGROUND/PURPOSE: The signaling mechanisms through which endothelin (ET)-1 induces hyperplasia of the renal tubular epithelium are largely unknown. METHODS: These mechanisms were explored using ETB-overexpressing opossum kidney (OKP) cells as a model system. RESULTS: ET-1 (10 nM) induced a 10-fold increase in c-jun mRNA abundance within 30 minutes and an 8-fold increase in extracellular signal-regulated kinase (ERK) 1/2 activity within 5-10 minutes in these cells. ERK1/2 phosphorylation in response to ET-1 was suppressed by ETB-receptor blockade or by treatment with an MAPK kinase (MEK) inhibitor. MEK1/2 activity increased 8-fold within 5 minutes of ET-1 treatment. Additionally 2-fold increases in cyclin D1 expression and retinoblastoma (RB) gene product phosphorylation were observed within 4 hours of treatment. CONCLUSION: Binding of ET-1 to the ETB receptor of ETB-overexpressing OKP cells is proposed to signal proliferation of these cells through rapid activation of mitogen-activated protein kinases, increased c-jun expression, modulation of cyclin D1 activity, and increased RB phosphorylation.

Down-regulation of D2 dopamine receptor and increased protein kinase Cmu phosphorylation in aldosterone-producing adenoma play roles in aldosterone overproduction.

CONTEXT: The mechanism associated with the overproduction of aldosterone by aldosterone-producing adenomas (APA) is unknown. OBJECTIVE: The objective of the study was to explore the role of the D2 dopamine receptor (D2R) on aldosterone synthesis and secretion and clarify the clinical importance of this role on aldosterone overproduction in APA. RESULTS: D2R expression in APA was examined in 24 patients and was much less than that in the nontumorous adrenal cortex. D2R mRNA levels in APA were inversely correlated with CYP11B2 mRNA levels and the patient's plasma aldosterone concentration. Angiotensin II (AII)-stimulated aldosterone secretion and CYP11B2 mRNA expression in human adenocarcinoma cells (H295R) was attenuated by the D2 agonist, bromocriptine (BMC). BMC selectively attenuated AII-induced protein kinase C (PKC)-mu phosphorylation and its translocation to the cell membrane. PKCmu-specific short-hairpin RNA significantly decreased AII-induced CYP11B2 mRNA expression and aldosterone secretion. BMC also attenuated the AII-induced increase in cytoplasmic calcium, partially through an inhibition of cytoplasmic inositol 1,4,5 triphosphate production. Despite similar total PKCmu levels in APA and the nontumorous adrenal cortex, expression of phosphorylated PKCmu in APA was much higher. CONCLUSION: This is the first study to demonstrate that the D2R modulated aldosterone secretion and synthesis through a specific attenuation of PKCmu activity, as well as the intracellular calcium level. Down-regulation of the D2R in APA, in turn, increased PKCmu activity and led to overproduction of aldosterone in affected patients. The D2R may thus serve as a potential treatment target for primary aldosteronism.

Anatomy instruction in medical schools: connecting the past and the future.

Anatomy curriculum has changed dramatically around the world since the 1960s. These changes include the reduction of course hours, the abandonment of cadaver dissection, the use of problem-based learning, application of other teaching modalities such as prosected specimens, models, radiographic images, computer simulations, and the introduction of humanity and death education. This article discusses the controversies in anatomy curriculum in Western countries, including the rationale for those changes, and the opinions of the objections.

Peritoneal fibrosing syndrome: pathogenetic mechanism and current therapeutic strategies.

Peritoneal dialysis (PD) has been established as a main renal replacement therapy for approximately 20 years. However, long-term peritoneal exposure to high glucose and other unphysiologic contents in the PD solution may potentiate the development of peritoneal fibrosing syndrome (PFS) in PD patients. PFS is composed of a wide spectrum of peritoneal alterations, which has been observed in PD patients. Molecular studies have shown that the fibrogenic effect of peritoneal mesothelial cells and the accompanying accumulation of extracellular matrix in the peritoneum are key events leading to PFS. In this review, we highlight the impact of PFS and its pathogenetic factors, including bioincompatible PD solution, multidisciplinary inflammatory mediators, and stimulatory cytokines in the peritoneal cavity. Current therapeutic strategies based on both clinical and basic evidence for the prevention or treatment of PFS are also reviewed.

Vasodilator agents inhibit opossum kidney proximal tubular cell growth.

Renal tubule cell hyperplasia and hypertrophy have been regarded as antecedents of tubulointerstitial fibrosis. Dipyridamole and pentoxifylline inhibit rat glomerular mesangial cell growth. The purpose of this study was to examine the effects of pentoxifylline and other vasodilators on opossum kidney proximal tubular (OKP) cell growth. A modified 3-(4,5-dimethylthiazil-2-yl)-2,5-diphenyltetrazolium bromide assay was used to evaluate cell proliferation in OKP cells. Pentoxifylline caused a dose-dependent inhibition of serum-stimulated proliferation of OKP cells: inhibition was 17%, 25%, and 37% at 0.03 mg/mL, 0.1 mg/mL, and 0.3 mg/mL, respectively. Dipyridamole 1.7 mg/L and 6 mg/L resulted in 34% and 46% inhibition, respectively. Verapamil 10(-6) M, 10(-5) M, and 10(-4) M resulted in 34%, 46%, and 67% inhibition, respectively. These inhibitory effects may contribute to renal protection or retardation of renal disease progression.

YC-1-inhibited proliferation of rat mesangial cells through suppression of cyclin D1-independent of cGMP pathway and partially reversed by p38 MAPK inhibitor.

This study was designed to investigate the effect of 1-benzyl-3-(5'-hydroxymethyl-2'-furyl) indazole (YC-1), a guanylate cyclase activator, upon the proliferation of rat mesangial cells and its underlying mechanism. YC-1 inhibited cell proliferation and DNA synthesis in a dose- and time-dependent manner. Flow cytometry cell-cycle studies revealed that YC-1 prevented the entry of cells from G1 into S phase. The expression of cyclin D1 and the kinase activity of cyclin D1/cyclin-dependent kinase (CDK)4 were lower within YC-1-treated cells, revealed by Western blotting, Northern blotting and kinase assays. YC-1 did not increase the intracellular cGMP concentration in mesangial cells. Inhibitors of soluble guanylate cyclase, protein kinase G, or protein kinase A also did not reverse the inhibitory effect elicited by YC-1, while co-treatment with p38 mitogen-activated protein kinase (MAPK) inhibitor could partially reverse the suppressive effect. YC-1 inhibited proliferation of mesangial cells and induced cell-cycle arrest by the reduction of cyclin D1 synthesis and cyclin D1/CDK4 kinase activity. This effect acts partially through p38 MAPK signal transduction activation and is independent of cGMP-signaling pathways.

The renoprotective potential of pentoxifylline in chronic kidney disease.

Current interventions with proven efficacy, such as glycemic and blood pressure control, dietary protein restriction, and angiotensin II blockade, slow the progression of chronic kidney disease (CKD); however, whether long-term cessation of CKD progression is possible remains unclear. Because of the pathogenetic complexity of this condition, multidrug interventions with the least adverse effects should be investigated as the next step in attempts to stop CKD progression. Pentoxifylline, a non-selective phosphodiesterase inhibitor with indiscernible toxicity, exerts potent inhibitory effects against cell proliferation, inflammation, and extracellular matrix accumulation, all of which play important roles in CKD progression. Pentoxifylline monotherapy markedly reduces proteinuria in patients with membranous nephropathy. Moreover, limited human studies have proven pentoxifylline efficacy in reducing proteinuria in patients with diabetes receiving angiotensin-converting enzyme inhibitors, and in patients with nephrotic syndrome secondary to lupus nephritis despite immunosuppressive therapy. Further clinical trials are necessary to examine whether pentoxifylline can improve renal outcomes in patients receiving interventions of proven efficacy.

Plasma interleukin-18 levels in chronic renal failure and continuous ambulatory peritoneal dialysis.

BACKGROUND/AIMS: Based on associations of interleukin (IL)-18 with chronic inflammation, we investigated IL-18, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in patients with chronic renal failure (CRF) and patients undergoing continuous ambulatory peritoneal dialysis (CAPD). METHODS: Plasma was evaluated by ELISA methodology in 15 healthy controls, 27 CRF and 15 CAPD patients. RESULTS: Plasma IL-18 levels in CRF (572.5 +/- 41.9 pg/ml) or CAPD (479.2 +/- 47.4 pg/ml) were significantly higher than normal (263.6 +/- 20.0 pg/ml), but there was no difference in IL-18 between CRF and CAPD patients. The IL-18 concentration negatively correlated with creatinine clearance (Ccr). However, the duration of dialysis, normalized protein nitrogen appearance, weekly Ccr, and Kt/V(urea) were not correlated with plasma IL-18 in CAPD. The plasma IL-18 concentration was positively correlated with TNF-alpha but not with IL-6 in renal failure patients with or without CAPD. CONCLUSION: Uremia is the principal origin of increased plasma IL-18 in these patients. Increased IL-18 levels may be associated with Th1 differentiation and elevated TNF-alpha.

The physiological impact of wearing an N95 mask during hemodialysis as a precaution against SARS in patients with end-stage renal disease.

BACKGROUND AND PURPOSE: Most patients with end-stage renal disease (ERSD) visiting our hospital for hemodialysis treatment during the SARS outbreak wore an N95 mask. Data on the physiological stress imposed by the wearing of N95 masks remains limited. This study investigated the physiological impact of wearing an N95 mask during hemodialysis (HD) on patients with ESRD. METHODS: ESRD patients who received regular HD at National Taiwan University Hospital between April to June 2003 were enrolled. Each patient wore a new N95 mask (3M Model 8210) during HD (4 hours). Vital signs, clinical symptoms and arterial blood gas measured before and at the end of HD were compared. RESULTS: Thirty nine patients (23 men; mean age, 57.2 years) were recruited for participation in the study. Seventy percent of the patients showed a reduction in partial pressure of oxygen (PaO2), and 19% developed various degrees of hypoxemia. Wearing an N95 mask significantly reduced the PaO2 level (101.7 +/- 12.6 to 92.7 +/- 15.8 mm Hg, p = 0.006), increased the respiratory rate (16.8 +/- 2.8 to 18.8 +/- 2.7/min, p < 0.001), and increased the occurrence of chest discomfort (3 to 11 patients, p = 0.014) and respiratory distress (1 to 17 patients, p < 0.001). Baseline PaO2 level was the only significant predictor of the magnitude of PaO2 reduction (p < 0.001). CONCLUSION: Wearing an N95 mask for 4 hours during HD significantly reduced PaO2 and increased respiratory adverse effects in ESRD patients.

Dual regulation of tumor necrosis factor-alpha-induced CCL2/monocyte chemoattractant protein-1 expression in vascular smooth muscle cells by nuclear factor-kappaB and activator protein-1: modulation by type III phosphodiesterase inhibition.

Monocyte/macrophage infiltration to the subendothelial space of arterial wall is a critical initial step in atherogenesis, in which CC chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1) is thought to play a key role. This study investigated the effectiveness of phosphodiesterase inhibitors, including the nonselective pentoxifylline (PTX) and the selective type III (cilostamide) and type IV (denbufylline) inhibitors, on cytokine-induced CCL2/MCP-1 production in cultured rat vascular smooth muscle cells (VSMCs), and the signal transduction mechanisms whereby they act. Our results showed that tumor necrosis factor (TNF)-alpha induced a marked increase in CCL2/MCP-1 production in dose- and time-dependent manners. 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126) [both inhibitors of p42/44 mitogen-activated protein kinase (MAPK) kinase], and anthra[1hyphen]9-cd]pyrazol-6(2H)-one (SP600125) [an inhibitor of c-Jun NH(2)-terminal kinases (JNKs)] attenuated TNF-alpha-induced CCL2/MCP-1 production, without affecting I-kappaBalpha degradation or p65/nuclear factor-kappaB (NF-kappaB) nuclear translocation. PD98059 abolished TNF-alpha-activated p42/44 MAPK phosphorylation and c-Fos up-regulation, whereas SP600125 inhibited TNF-alpha-activated JNK and c-Jun phosphorylation. The NF-kappaB inhibitor carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132) attenuated TNF-alpha-induced CCL2/MCP-1 production in the presence of increased phospho-JNK and phospho-c-Jun levels. When SP600125 was added simultaneously, MG132 completely inhibited TNF-alpha-induced CCL2/MCP-1 production. Finally, the pretreatment of VSMCs with PTX or cilostamide, but not denbufylline, reduced TNF-alpha-induced CCL2/MCP-1 production, which was preceded by attenuation of p65/NF-kappaB nuclear translocation, p42/44 MAPK, and JNK-c-Jun phosphorylation, and c-Fos up-regulation. These data indicate that TNF-alpha-stimulated CCL2/MCP-1 production in rat VSMCs is dually regulated by activator protein-1 (AP-1) and NF-kappaB pathways, and inhibition of type III phosphodiesterase contributes substantially to the suppressive effect of PTX on CCL2/MCP-1 production via down-regulation of AP-1 and NF-kappaB signals.

Expression of CX3CL1/fractalkine by mesangial cells in vitro and in acute anti-Thy1 glomerulonephritis in rats.

BACKGROUND: Mesangial cells (MCs) can promote glomerular macrophage accumulation in glomerulonephritis through production of a variety of chemokines. This study investigated the potential of MCs to synthesize CX3CL1/fractalkine, a CX3C chemokine, both in vitro and in acute anti-Thy1 glomerulonephritis in rats. METHODS: Anti-Thy1 glomerulonephritis was induced in Wistar rats by a single injection of mouse anti-rat Thy1.1 antibody intravenously. Glomerular mRNAs for CX3CL1/fractalkine, CCL2/monocyte chemoattractant protein (MCP)-1, and their cognate receptors, CX3CR1 and CCR2, were determined by northern blot analysis or reverse-transcription polymerase chain reaction. CX3CL1/fractalkine mRNA and protein expression in vivo was localized by in situ hybridization and immunohistochemistry. Monocytes/macrophages and activated MCs were detected by immunohistochemistry. Regulation of CX3CL1/fractalkine expression in cultured MCs was determined by northern and western blot analysis. RESULTS: After induction of anti-Thy1 disease, glomerular CX3CL1/fractalkine mRNA was significantly up-regulated, peaking at 2 h and sustaining into day 5 of the nephritis. A corresponding increase in urinary CX3CL1/fractalkine protein was evident after day 1 of the nephritis, but became more prominent during the MC proliferative phase (days 3-5). Meanwhile, induction of glomerular CCL2/MCP-1 mRNA and urinary CCL2/MCP-1 protein occurred within 24 h, and was barely detectable after day 3 of the nephritis. Urinary CCL2/MCP-1, but not CX3CL1/fractalkine, correlated with glomerular macrophage accumulation (r = 0.936, P<0.01) and glomerular CCR2 mRNA expression (r = 0.965, P<0.01). In contrast, only urinary CX3CL1/fractalkine coincided temporally to glomerular mRNA for CX3CR1 (r = 0.809, P < 0.01). Combined in situ hybridization and immunohistochemistry revealed that activated MCs were a major source for CX3CL1/fractalkine mRNA and protein during days 3-5 of the nephritis. Incubation of cultured MCs with tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, platelet-derived growth factor (PDGF)-AB or basic fibroblast growth factor (bFGF) significantly up-regulated CX3CL1/fractalkine mRNA and protein expression. This cytokine- and growth factor-stimulated CX3CL1/fractalkine expression could be abolished by the nuclear factor-kappaB inhibitors, curcumin and MG132. CONCLUSIONS: Our data demonstrate that activated MCs are a source for the augmented glomerular CX3CL1/fractalkine expression during the proliferative phase of acute anti-Thy1 glomerulonephritis. Up-regulation of MC CX3CL1/fractalkine by TNF-alpha, IL-1beta, PDGF-AB and bFGF is mediated, at least in part, via the nuclear factor-kappaB signalling pathway. The differential expression of CCL2/MCP-1 and CX3CL1/fractalkine may sequentially recruit distinct subsets of monocytes to the glomerulus during acute anti-Thy1 glomerulonephritis.

Pentoxifylline inhibits platelet-derived growth factor-stimulated cyclin D1 expression in mesangial cells by blocking Akt membrane translocation.

Pentoxifylline (PTX) is a potent inhibitor of mesangial cell proliferation, but its underlying mechanism is poorly understood. Here, we demonstrate that in platelet-derived growth factor (PDGF)-stimulated mesangial cells, PTX causes G1 arrest by down-regulation of cyclin D1 expression, which subsequently attenuates Cdk4 activity. In vivo, PTX similarly reduces cyclin D1 expression in mesangial cells of rats with acute Thy1 glomerulonephritis. The mechanism by which PTX reduces cyclin D1 is also investigated. PTX blocks Akt but not phosphatidylinositol 3-kinase (PI3K) activation in response to PDGF and abrogates cyclin D1 induction by PI3K, suggesting an effect of PTX on Akt itself. Indeed, PTX is capable of blocking the membrane translocation of Akt, and enforced targeting of Akt to cell membrane prevents the inhibition of Akt and cyclin D1 by PTX. Because PTX is known to increase intracellular cAMP levels by inhibiting phosphodiesterase, the role of protein kinase A (PKA) in these events is investigated. The PKA antagonist N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89) abolishes cell proliferation effects of PTX and restores cyclin D1 expression as well as Akt membrane translocation and activation by PDGF, whereas dibutyryl cAMP and forskolin recapitulate the functions of PTX in mesangial cells. In conclusion, our results indicate that PTX, acting through PKA, interferes with PDGF signaling to Akt activation by blocking Akt membrane translocation, thereby inhibiting cyclin D1 expression and mesangial cell proliferation.