Chronic kidney disease promotes chronic inflammation in visceral white adipose tissue.

White adipose tissue plays an important role in the development of metabolic disturbance, which is a common feature in patients with chronic kidney disease (CKD). The effect of CKD on white adipose tissue remains poorly appreciated. Here, we evaluated the inflammatory potential of visceral white adipose tissue in a rat model of CKD. The results showed that production of proinflammatory cytokines and infiltration of macrophage in the tissue were increased significantly in CKD rats compared with sham rats. Moreover, the primary adipocytes and stromal vascular fraction under the condition of CKD could trigger the inflammatory response in each other. Free fatty acid induced robust inflammatory response in ex vivo peritoneal-derived macrophages from CKD rats, which was associated with reduced activity of silent information regulator T1 (SIRT1). Improvement of SIRT1 activity by an activator could alleviate free fatty acid-induced inflammatory response in the macrophages and inflammation in the white adipose tissue. Moreover, oxidative stress occurred in the tissue and linked with the reduced activity of SIRT1 in macrophages and enhanced release of free fatty acid in the tissue. We thus identified CKD as a risk factor for chronic inflammation in white adipose tissue. These observations might open up new therapeutic strategies for metabolic disturbance in CKD via the modulation of adipose tissue-related pathways.

[Analysis of pregnancy outcomes in 66 patients with systemic lupus erythematosus].

OBJECTIVE: To analyze the outcomes of pregnancies in women with systemic lupus erythematosus (SLE) and the risk factors affecting the outcomes. METHODS: The data of SLE patients with pregnancy admitted from October, 2006 and September, 2015 were analyzed for assessing the maternal and fetal outcomes and complications. Their risk factors affecting the outcomes of the pregnancies were analyzed. RESULTS: The 66 SLE patients (69 pregnancies) had a mean age at SLE diagnosis of 22.9 ∓ 5.1 years with a mean duration of SLE of 4.1∓3.6 years before pregnancy. Forty-five (65.2%) of the patients received oral medication for SLE treatment during pregnancy, and 44 (63.8%) were treated with prednisone and 19 (27.5%) were treated with hydroxychloroquine. The highest SLEDAI score was 6.8∓7.4 during pregnancy. The patients with moderate-to-severe disease activity had a higher rate of fetal loss (12 [54.5%] vs 12 [25.5%]) with a significantly lower birth weight of the newborns than those with remittent or mild disease (2073.0∓ 778.7 vs 2817.8∓533.7 g, P<0.05). The patients with moderate-to-severe disease activity also had higher rates of new-onset SLE (9 [40.9%] vs 6 [12.8%]), hypertension (12 [54.5%] vs 3 [6.4%]), active lupus nephritis (22 [100%] vs 4 [8.5%]), pneumonia (5 [22.7%] vs 2 [4.3%]), and renal insufficiency (8 [36.4%] vs 2 [4.3%]) compared with patients with remittent and mild disease (P<0.05). Active lupus nephritis (OR=6.10,95%CI: 1.43-25.96) was a significant predictor of adverse outcomes of the pregnancies. CONCLUSION: Fetal loss and maternal complications are common in patients with SLE in relation with the disease activity. Active lupus nephritis is a predictor for poor outcomes of pregnancies in SLE patients.

Enhanced M1 and Impaired M2 Macrophage Polarization and Reduced Mitochondrial Biogenesis via Inhibition of AMP Kinase in Chronic Kidney Disease.

BACKGROUND: Macrophage polarization plays a pivotal role in the process of inflammation which is common in chronic kidney disease (CKD). Macrophages polarization under the condition of CKD remains poorly understood. Here we tested the hypothesis that CKD promotes macrophage M1 polarization. METHODS: A rat model of CKD was established by reduced renal mass (RRM). Polarization of macrophages was induced in ex vivo macrophages from RRM rats and cultured ones under the condition of uremic serum. The markers were evaluated by RT-PCR, western blot, and flow cytometer. RESULTS: Our data showed that macrophages from RRM rats displayed enhanced M1 and impaired M2 polarization as revealed by increased M1 markers (tumor necrosis factor α, IL-6, IL-12p40, nitric oxide) and decreased M2 markers (IL-10, CD206, arginase activity) in response to LPS and IL-4 induction, respectively. Treatment with uremic sera in peritoneal and bone marrow derived macrophages from normal rats led to similar results. Moreover, macrophages from RRM rats and cultured under the condition of uremic sera had reduced mitochondrial biogenesis. The disturbed macrophage polarization and mitochondrial biogenesis were accompanied by reduced activity of adenosine monophosphate-activated protein (AMP)-activated kinase (AMPK). Enhancing activation of AMPK restored mitochondrial biogenesis and M2 macrophage polarization. CONCLUSION: These observations suggest that CKD disturbs macrophage polarization and mitochondrial biogenesis through inhibition of AMPK. This might provide a novel therapeutic strategy for intervention of chronic inflammation in CKD.

The effect of inhibition of endoplasmic reticulum stress on lipolysis in white adipose tissue in a rat model of chronic kidney disease.

AIM: Lipolysis in fat tissue plays an important role in the development of metabolic disturbances, a characteristic feature of chronic kidney disease (CKD). In the present study, we tested the hypothesis that the inhibition of endoplasmic reticulum (ER) stress could alleviate lipolysis in white adipose tissue in a rat model of CKD. METHODS: A rat model of CKD was established by a method of reduced renal mass (RRM). Lipolysis was measured as the release of glycerol in ex vivo fat pads and cultured primary adipocytes. The activity of lipases and markers of ER stress were measured by Western blotting and immunoprecipitation. RESULTS: Our data showed that lipolysis in visceral white adipose tissue was increased in RRM rats compared with control rats. In addition, increased phosphorylation of hormone-sensitive lipase (HSL) and binding of adipose triglyceride lipase (ATGL) to comparative gene identification-58 (CGI-58) protein were observed in the RRM rats. The phosphorylation of ER stress markers, including IRE1α, PERK, and eukaryotic initiation factor (eIF) 2α, and the expression of ER stress marker 78 kDa glucose-regulated protein (GRP78) were significantly increased in RRM rats. Treatment with an inhibitor of ER stress partially but significantly alleviated lipolysis, and this alleviation was accompanied by reduced binding of ATGL to CGI-58. CONCLUSION: Our results showed that enhanced lipolysis and ER stress occurred in visceral white adipose tissue in a rat model of CKD. Moreover, inhibition of ER stress significantly alleviated lipolysis. These findings suggest that ER stress is a potential therapeutic target for the metabolic disturbances associated with CKD.

Current pattern of Chinese dialysis units: a cohort study in a representative sample of units.

BACKGROUND: Understanding the characteristics of Chinese dialysis patients and the current practice trends is the first step to evaluate the association between practice pattern and outcome in these populations. In the present study, we evaluated the status of medical treatment and characteristic features of chronic dialysis patients in China. METHODS: Through a clustering sampling, we selected 9 centers from the largest dialysis facilities in 6 cities around China. All adult undergoing dialysis in the selected units were screened. A total of 2388 (1775 on hemodialysis (HD) and 613 on peritoneal dialysis (PD)) patients were finally enrolled. All data were collected at enrollment on the bases of review of medical records. RESULTS: In this cohort, 1313 (55.0%) were male. The mean age was 54 years old. The median time for dialysis was 26 months (12 - 51 months). Seventy-five percent of patients were on HD and 25.0% on PD. Among PD patients, about 21% patients did not receive dialysis adequacy. For HD patients, about 14.0% of them did not achieve dialysis adequacy when the target of kt/V was set as 1.2. Only 44.7% of patients achieved blood pressure target of 140/90 mmHg. About 60% of patients did not reach the hemoglobin target of 110 g/L even though 85.0% of them were treated with erythropoietin. In addition, 48.5% of the patients had uncontrolled mineral metabolism revealed by the high calcium-phosphate product. Compared with HD patients, higher level of serum glucose, triglyceride, and total and low density lipoprotein cholesterol were more common in PD patients. CONCLUSIONS: This observational study suggests that many Chinese dialysis patients did not achieve the therapeutic target, particularly in blood pressure control, anemia correction, and mineral balance. PD patients were more likely to suffer metabolic disturbance.

Vascular insulin resistance related to endoplasmic reticulum stress in aortas from a rat model of chronic kidney disease.

Metabolic insulin resistance has been demonstrated in patients with nondiabetic chronic kidney disease (CKD), yet their vascular insulin signaling remains poorly understood. Here we tested the hypothesis that vascular insulin signaling was impaired and related with endoplasmic reticulum (ER) stress in aortas from the reduced renal mass (RRM) model of CKD. The activity of insulin signaling and markers of ER were determined in aortas from rats with RRM and cultured human umbilical vein endothelial cells. Tyrosine phosphorylation of insulin receptor-ß and insulin receptor substrate (IRS)-1 and phosphorylation of protein kinase B and endothelial nitric oxide synthase were all decreased in aorta from RRM rats, whereas serine phosphorylation of IRS-1, a marker of insulin resistance, was increased. In addition, nitric oxide generation and insulin-mediated vasorelaxation were decreased in aortas from RRM rats. Insulin signaling in cultured vascular endothelial cells was impaired by induction of ER stress and was restored in aortas of RRM rats by inhibition of ER stress. Taken together, rats with RRM had vascular insulin resistance that was linked to ER stress. This identified vascular insulin resistance and ER stress as a potential therapeutic target for cardiovascular complications in patients with CKD.

Changes in pathological pattern and treatment regimens based on repeat renal biopsy in lupus nephritis.

BACKGROUND: Relapses occur frequently in patients with lupus nephritis. Renal biopsy is the gold standard for assessing renal activity and hence guiding the treatment. Whether repeat renal biopsy is helpful during flares of lupus nephritis remains inconclusive. In the present study, we retrospectively reviewed the patients with lupus nephritis who had more than one renal biopsy with the hope to find the clinical value of repeat biopsy. METHODS: Patients who had a diagnosis of lupus nephritis and two or more renal biopsies were selected from the database of the patient pathology registration at this renal division. Renal biopsy was evaluated according to the International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification of lupus nephritis. The pathological patterns and treatment regimens were analyzed after a repeat biopsy. RESULTS: We identified 44 systemic lupus erythematosus patients with serial renal biopsies. In total, there were 94 renal biopsies. Overall, the pathological transition occurred in 64% instances according to the ISN/RPS class. When the transition was analyzed according to proliferative, membranous or mix lesions, it showed different profile: 35% in patients with proliferative lesion, 23.5% patients with mix lesions, 100% in patients with pure membranous lesion. The pathological transition could not be predicted by any clinical characteristics. After the repeat renal biopsy, 34% of patients had a change in their treatment regimens. CONCLUSIONS: The pathological conversion was very prevalent in patients with lupus nephritis. However, the transitions became less prevalent when they were analyzed according to pure membranous, proliferative, and mix lesion. Repeat biopsy might be helpful to avoid unnecessary increased immunosuppression therapy.

VEGF ameliorates tubulointerstitial fibrosis in unilateral ureteral obstruction mice via inhibition of epithelial-mesenchymal transition.

AIM: Vascular endothelial growth factor (VEGF) has been shown to be a survival factor for renal tubular epithelial cells. In the present study, we investigated whether administration of VEGF ameliorates tubulointerstitial fibrosis in a mouse model of unilateral ureteral obstruction (UUO). METHODS: Thirty-six male CD-1 mice were randomly divided into three groups: sham-operation, UUO and UUO+VEGF group. VEGF (50 µg/kg) was subcutaneously injected twice daily from d 1 to d 14. Mice in each group were killed at d 3, 7, or 14 after the operation, and the tubulointerstitial fibrosis was histopathologically evaluated. Human proximal tubular epithelial cells (HK-2) were used for in vitro study. The expression levels of α-SMA, E-cadherin, TGF-ß1, CTGF, and BMP-7 in the kidney were determined using Western blot and RT-PCR. RESULTS: In the UUO mice, the degree of interstitial fibrosis was dramatically increased in a time-dependent manner. At d 3, 7, and 14, both the mRNA and protein expression levels for α-SMA, TGF-ß1, and CTGF were significantly upregulated, whereas those for E-cadherin and BMP-7 were significantly downregulated. At d 3 and 7, VEGF treatment significantly reduced interstitial fibrosis and the expression levels for α-SMA, TGF-ß1, and CTGF, while significantly increased the expression of E-cadherin and BMP-7, as compared with the UUO mice. At d 14 after operation, no significant differences were observed in the expression of the examined markers between VEGF-treated mice and UUO mice, with the exception of CTGF. In HK-2 cells, VEGF blocked TGF-ß1-induced α-SMA and vimentin expression and restored E-cadherin expression in a dose-dependent manner. CONCLUSION: VEGF may ameliorate renal tubulointerstitial fibrosis at the early stage in UUO mice. This effect may be related to inhibition of VEGF on renal tubular epithelial-mesenchymal transition (EMT).

Advanced oxidation protein products induce inflammatory response and insulin resistance in cultured adipocytes via induction of endoplasmic reticulum stress.

Accumulation of advanced oxidation protein products (AOPPs) is prevalent in metabolic syndrome and type 2 diabetes. Adipocyte dysfunction has been recognized as a link between these conditions. To examine the effect of AOPPs on adipocyte perturbation, 3T3-L1 adipocytes were treated with increased levels of AOPPs as seen in these conditions. Exposure of adipocytes to AOPPs induced overexpression of tumor necrosis factor α and interleukin-6. This inflammatory response was completely blocked by nuclear factor-κB inhibitor SN50. AOPPs challenge also impaired insulin signaling, which was partly prevented by SN50. Treatment with AOPPs triggered endoplasmic reticulum (ER) stress, revealed by phosphorylation of PKR-like eukaryotic initiation factor 2α kinase, eukaryotic translational initiation factor 2α, inositol-requiring enzyme 1 and c-jun N-terminal kinase, and by overexpression of glucose regulated protein 78. AOPPs-induced ER stress was mediated by reactive oxygen species (ROS) generated by activation of NADPH oxidase since it was prevented by NADPH oxidase inhibitors or ROS scavenger. Treating the cells with inhibitors of NADPH oxidase or ER stress could completely abolish AOPPs-induced overexpression of adipocytokines and insulin resistance, suggesting that AOPPs induced adipocyte perturbation probably through induction of ROS-dependent ER stress. Our data identified AOPPs as a class of important mediator of adipocyte perturbation. Accumulation of AOPPs might be involved in adipocyte dysfunction as seen in metabolic syndrome and type 2 diabetes.

Advanced oxidation protein products inhibit differentiation and activate inflammation in 3T3-L1 preadipocytes.

Accumulation of advanced oxidation protein products (AOPPs) is prevalent in metabolic syndromes, a condition with impaired preadipocytes differentiation. In the present study, we tested the hypothesis that AOPPs disturb preadipocyte differentiation. Exposure of 3T3-L1 preadipocytes to increased levels of AOPPs inhibited accumulation of intracellular triglyceride and decreased the expression of the essential markers of matured adipocytes, such as adipocyte fatty-acid-binding protein (aP2), CAAT/enhancer-binding protein (C/EBP)-alpha, and peroxisome proliferator-activated receptor (PPAR)-gamma, in response to standard adipogenic induction. Inhibitory effects of AOPPs on preadipocytes differentiation was time sensitive, which occurred at the early stage of differentiation. In the presence of AOPPs, induction of preadipocytes differentiation resulted in upregulated expression of C/EBP homologous protein (CHOP) and CUG-Triplet repeat-binding protein (CUGBP), two important inhibitors of preadipocytes differentiation. In addition, treatment with AOPPs increased abundance of C/EBP-beta-liver enriched inhibitory protein (C/EBP-beta-LIP), a truncated C/EBP-beta isoform without adipogenic activity. Moreover, AOPPs-treated preadipocytes expressed a macrophage marker F4/80 and overexpressed tumor necrosis factor-alpha and interleukin-6 via nuclear factor-kappaB (NF-kappaB)-dependent pathway. However, blocking inflammation with NF-kappaB inhibitor failed to improve AOPPs-induced inhibition of preadipocytes differentiation. These data suggest that accumulation of AOPPs may inhibit differentiation of preadipocytes and activate inflammation in these cells. This information might have implication for understanding the impairment of preadipocytes differentiation and fat inflammation seen in metabolic syndrome.

Optimal dose of angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker for renoprotection.

Angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II type 1 receptor blockers (ARB) have become the cornerstone in the treatment of chronic kidney disease (CKD), as numerous lines of evidence have shown that these agents have a blood pressure lowing independent anti-proteinuric effect. However, despite the benefits of ACEI or ARB therapy, a substantial proportion of patients still experience renal morbidity and mortality. Considering the prognostic impact of proteinuria reduction, it is currently assumed that titration of ACEI or ARB for optimal anti-proteinuric effect would be a logical step towards improvement of renoprotection. Recent published studies, performed with higher than recommended doses of either ACEI or particularly ARB, suggest that the approach is associated with a further decrement in urinary protein excretion and probably improved renal outcome. Although most patients achieve their maximum benefit at standard doses, there is a residual group of patients who may do so at higher doses of renin-angiotensin system inhibitors. Because patients who would benefit from higher doses are not identifiable a priori, a titration process might be cogent in order to provide more robust anti-proteinuric benefit to such patients.

[Estrogen reduces the expressions of ryanodine receptor type 1 and Cav1.3 L-type calcium channel in the vaginal smooth muscle cells of rats].

OBJECTIVE: To determine the expressions of ryanodine receptor type 1 (RyR1) and Cav1.3 L-type calcium channel (Cav1.3) in the vaginal smooth muscle cells of castrated rats and investigate the correlation of RyR1 and Cav1.3 with estrogen in female sexual dysfunction. METHODS: Forty female SD rats of 8 weeks were randomly divided into Groups A (2-week sham operation), B (4-week sham operation), C (2-week castration) and D (4-week castration). Two and 4 weeks after surgery, the serum estradiol level was determined with the automated immunochemiluminescence system and the expressions of RyR1 and Cav1.3 in the vaginal smooth muscle were detected by immunohistochemistry and RT-PCR. Gray scale ratio was used to represent the mRNA expression levels of RyR1 and Car1.3, and the optical density value to denote their protein expression levels. RESULTS: Serum estradiol was significantly decreased in Group C ([0.210 +/- 0.026] nmol/L) as compared with A ([0.505 +/- 0.053] nmol/L) (P < 0.01), and so was it in Group D ([0.130 +/- 0.031] nmol/L) in comparison with B ([0.476 +/- 0.058] nmol/L) (P < 0.01). RyR1 and Cav1.3 were expressed in all groups. The mRNA expressions of RyR1 and Cav1.3 were significantly reduced in Group C (0. 680 +/- 0.073 and 0.580 +/- 0.043) as compared with A (0.950 +/- 0.064 and 0.870 +/- 0.019) (P < 0.01), as well as in Group D (0.220 +/- 0.032 and 0.190 +/- 0.020) in comparison with B (0.890 +/- 0.072 and 0.820 +/- 0.021) (P < 0.01). The protein expressions of RyR1 and Cav1.3 were significantly down-regulated in Group C (96.67 +/- 7.75 and 87.97 +/- 6.96) as compared with A (123.69 +/- 10.66 and 106.46 +/- 8.04) (P < 0.01), and so were they in D (86.45 +/- 8.16 and 69.43 +/- 8.30) in comparison with B (109.31 +/- 9.87 and 97.38 +/- 7.56) (P < 0.01). CONCLUSION: Both RyR1 and Cav1.3 were expressed in the vaginal smooth muscle cells of the rats, and estrogen might be involved in the regulation of female sexual reaction by acting on the expressions of RyR1 and Cav1.3.

Asymmetrical dimethylarginine triggers lipolysis and inflammatory response via induction of endoplasmic reticulum stress in cultured adipocytes.

Protein energy wasting, a state of decreased stores of body protein and fat, is a risk factor for mortality in advanced chronic kidney disease (CKD). Little is known about the mechanism underlying loss of fat in CKD. Accumulation of asymmetric dimethylarginine (ADMA) is prevalent in advanced CKD. Here we assessed the effect of ADMA on cellular perturbation in cultured 3T3-L1 adipocytes. Exposure of adipocytes to ADMA induced lipolysis and decreased perilipin A, with no alteration of lipases expression or activity. ADMA treatment also upregulated the expression of inflammatory adipocytokines via activation of nuclear factor-kappaB (NF-kappaB). Blocking the inflammatory responses with NF-kappaB inhibitor partly inhibited the ADMA-induced lipolysis. Furthermore, ADMA treatment triggered endoplasmic reticulum (ER) stress, revealed by phosphorylation of PKR-like eukaryotic initiation factor 2alpha kinase, eukaryotic translational initiation factor 2alpha, c-Jun NH2-terminal kinase, and overexpression of glucose-regulated protein 78. Treatment with ER stress inhibitor completely abolished the ADMA-induced lipolysis and inflammatory responses. Moreover, conditioned medium from the ADMA-treated adipocytes increased protein degradation in cultured C2C12 myotubes, suggesting that the ADMA-induced adipocyte perturbation may promote skeletal muscle proteolysis. These data suggest that elevated ADMA promoted the adipocyte perturbation through induction of ER stress, which might have implication for protein energy wasting in CKD.

Advanced oxidation protein products induce mesangial cell perturbation through PKC-dependent activation of NADPH oxidase.

Mesangial deposition of extracellular matrix (ECM) is a hallmark of several glomerular diseases including diabetic nephropathy. Accumulation of advanced oxidation protein products (AOPPs) has been found in diabetes and chronic kidney disease and linked to mesangial ECM deposition and progressive glomerulosclerosis in these disorders. Although emerging evidence implicates AOPPs as the renal pathogenic factors, the underlying mechanisms have not been investigated. Here, using cultured rat mesangial cells (MCs) as a model, we identify AOPPs as the important mediators for activation of MC NADPH oxidase. Exposure of MCs to AOPPs, through membrane-associated phosphorylation of PKCalpha, induced rapid phosphorylation of cytosolic p47(phox) and its membrane translocation, enhanced interaction of p47(phox) with the membrane components p22(phox) and Nox4, and increased expression of these key regulatory subunits of NADPH oxidase. Challenge with AOPPs triggered cytosolic superoxide generation, resulting in upregulation of fibronectin and collagen IV genes and proteins and overexpression of TGF-beta1 via a PKC-NADPH oxidase-dependent pathway, as these downstream events were blocked by the inhibitors of PKC, inhibitors of NADPH oxidase, or the cytosolic superoxide scavenger. These data provide new information for understanding the molecular basis underlying AOPP-induced MC perturbation and might be a central step toward development of new interventions.

Inhibition of tubulointerstitial fibrosis by pentoxifylline is associated with improvement of vascular endothelial growth factor expression.

AIM: Recent information indicates that pentoxifylline (PTX) has the ability to suppress inflammation and profibrotic cell proliferation. In this study, we investigated the effect of PTX on tubulointerstitial fibrosis and the expression of vascular endothelial growth factor (VEGF) in a rat model of obstructive nephropathy. METHODS: Wistar rats with left ureteral ligation were divided into control and PTX-treated groups. The histopathologic degree of tubulointerstitial fibrosis was scored with PAS and Masson-stained sections. The protein and mRNA for vascular endothelial growth factor (VEGF) were semiquantitatively measured with immunohistochemistry and RT-PCR. The protein for transforming growth factor beta1 (TGFbeta1) and hypoxia-induced factor 1 alpha (HIF-1alpha) was determined by Western blot. RESULTS: Compared with the control group, PTX treatment reduced fibrosis scores at d 7 and d 14 (P<0.05). The reduction was accompanied by inhibited expression of transforming growth factor-beta 1 (TGFbeta1), a key cytokine in tubulointerstitial fibrogenesis (P<0.01). Meanwhile, VEGF protein and mRNA in the kidney were increased in the PTX-treated group compared with the control group (P<0.01). PTX up-regulated expression of VEGF mRNA in a dose- and time-dependent manner in cultured HK-2 cells (P<0.01). However, expression of HIF-1alpha (a key transcription factor for VEGF gene expression) was unchanged by PTX treatment. PTX prolonged the half-life of VEGF mRNA by a 1.07-fold increase. CONCLUSIONS: PTX inhibited tubulointerstitial fibrosis in a rat model of obstructive nephropathy while preventing loss of VEGF. PTX up-regulated expression of VEGF mRNA through stabilization of its mRNA in cultured renal tubular epithelial cells.

1,25-Dihydroxyvitamin D improved the free fatty-acid-induced insulin resistance in cultured C2C12 cells.

BACKGROUND: Epidemiological evidence has indicated that vitamin D deficiency increased the risk of insulin resistance in metabolic syndrome. The present study was conducted to test the hypothesis that 1,25-dihydroxyvitamin D may improve the free fatty-acid (FFA)-induced insulin resistance in muscle cells. METHOD: The insulin resistance of muscle cell model was established by treatment of FFA in differentiated C2C12 cells. Glucose uptake of C2C12 myotubes was analysed by the 3H-labelled 2-deoxyglucose uptake assay. The diameter of myotubes was measured under the condition of glutaraldehyde-induced autofluorescense. Tyrosine phosphorylated insulin receptor substrate 1 (IRS-1) was measured by immunoprecipitation. Serine phosphorylated IRS-1 and protein kinase B (Akt), extracellular signal-related kinase (ERK), c-Jun amino-terminal kinases (JNK) as well as their phosphorylated form were analysed by Western blots. RESULTS: Compared with a vehicle-treated group, FFA treatment in myotubes was associated with 70.6% reduction in insulin-mediated uptake of glucose, a five-fold increase in serine phosphorylation of IRS-1, 76.9% decrease in tyrosine phosphorylation of IRS-1 and 81.8% decrease in phosphorylation of Akt. Supplement of 1,25-dihydroxyvitamin D improved the FFA-induced inhibition of glucose uptake in a dose- dependent (p < 0.001) and time-dependent manner (p < 0.01). This was accompanied by increase in tyrosine phosphorylation of IRS-1 and phosphorylated Akt and decrease in serine phosphorylation of IRS-1 (p < 0.001). 1,25-Dihydroxyvitamin D also inhibited the FFA-induced reduction in myotube diameter by 35.3% (p < 0.001). JNK phosphorylation was reduced by 126.7% with treatment of 1,25-dihydroxyvitamin D (p < 0.001). 1,25-Dihydroxyvitamin D had no effect on FFA-induced ERK phosphorylation (p = 0.84). CONCLUSION: 1,25-Dihydroxyvitamin D improved the FFA-induced insulin resistance in muscle cells.

[Effect of bone morphogenetic protein-7 on monocyte chemoattractant protein-1 induced epithelial-myofibroblast transition and TGF-beta1-Smad 3 signaling pathway of HKC cells].

OBJECTIVE: To examine the effect of Bone Morphogenetic protein-7 (BMP-7) on Monocyte chemoattractant protein-1 (MCP-1) induced epithelial-myofibroblast transition (EMT) in cultured renal proximal tubular cells (HK-2) and the relationship between TGF-beta1-smad 3 expressions and MCP-1 induced EMT. METHODS: The cultured HK-2 cells were divided into six groups: a, negative control, b, treated with TGF-beta1 (5 ng/ml) as positive control, c, treated with MCP-1 (0.1, 1, 10, 50 ng/ml), d, treated with BMP-7 (0.1, 1, 10, 50 ng/ml), e. co-treated with MCP-1 (1 ng/ml) and MCP-1 neutralized antibody (1 ng/ml), f. co-treated with MCP-1 (1 ng/ml) and BMP-7 (50 ng/ml). alpha-Smooth Muscle Actin (alpha-SMA) mRNA expression of HK-2 cells was assessed with RT-PCR. Secretion of type I collagen was assessed with RT-PCR and ELISA, respectively. TGF-beta1 and Smad 3 expressions were assessed with Western blot. RESULTS: alpha-SMA mRNA expression significantly increased in HK-2 cells treated with MCP-1 (0.1, 1 ng/ml) compared with negative controls (5.97 +/- 0.35, 23.36 +/- 1.37 vs. 0.59 +/- 0.38, P < 0.01). alpha-SMA mRNA expression of HK-2 cells concomitantly treated with MCP-1 neutralized antibody or BMP-7 (50 ng/ml) and MCP-1 (1 ng/ml) significantly decreased than that in cells treated with MCP-1 (1 ng/ml) alone (1.93 +/- 0.34, 13.59 +/- 0.38 vs. 36.36 +/- 1.37, P < 0.01). Secretion of type I collagen of the cells treated with MCP-1 (0.1, 1 ng/ml) markedly increased compared with negative control (1751 +/- 34, 1876 +/- 45 vs. 1450 +/- 62; P < 0.01). The secretion of type I collagen of the supernatant were also significantly lower than that in cells treated with MCP-1 (1 ng/ml) alone (1462 +/- 56, 1596 +/- 34 vs. 1876 +/- 45, P < 0.05). The expression of TGF-beta1 and Smad 3 of HK-2 cells treated with MCP-1 (1 ng/ml) were markedly higher than that of negative controls, respectively (36.31 +/- 1.37 vs. 0.75 +/- 0.16, P < 0.01; 56.98 +/- 2.61 vs. 23.05 +/- 1.82, P < 0.01). The expressions of TGF-beta1 and Smad 3 in HK-2 cells treated concomitantly with MCP-1 neutralized antibody or BMP-7 (50 ng/ml) and MCP-1 (1 ng/ml) were markedly decreased than that treated with MCP-1 alone, respectively. (4.61 +/- 0.74, 23.74 +/- 2.14 vs. 36.31 +/- 1.37, P < 0.01; 19.63 +/- 1.65, 37.06 +/- 1.82 vs. 56.98 +/- 2.61, P < 0.01). The expressions of TGF-beta1 and Smad 3 in HK-2 cells treated concomitantly with MCP-1 neutralized antibody or BMP-7 (50 ng/ml) and MCP-1 (1 ng/ml) were markedly decreased than that treated with MCP-1 alone, respectively. (4.61 +/- 0.74, 23.74 +/- 2.14 vs. 36.31 +/- 1.37, P < 0.01; 19.63 +/- 1.65, 37.06 +/- 1.82 vs. 56.98 +/- 2.61, P < 0.01). CONCLUSIONS: The results documented that MCP-1 may induce EMT of HK-2 cells in vitro, and this effect is related to up-regulated expression of TGF-beta1 and Smad 3. BMP-7 may partially inhibit MCP-1-induced EMT and this effect is related to the downregulated expression of TGF-beta1 and Smad 3 of the cells. The results also suggest that MCP-1 induced EMT may involve the TGF-beta1-independent pathway of the cells.

[Altered expression of vascular endothelial growth factor and its receptors in transdifferentiated human proximal tubular epithelial cells induced by transforming growth factor beta1].

OBJECTIVE: To examine the expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFR1, VEGFR2) in transdifferentiated human proximal tubular epithelial (HK-2) cell induced by transforming growth factor beta1 (TGFbeta1). METHODS: The transdifferentiation of HK-2 cells was detected by evaluation of expression of alpha-SMA by cytoimmunochemistry and RT-PCR. The VEGF mRNA was evaluated with RT-PCR. The secreted VEGF in the culture media was measured with ELISA. The cellular VEGF, VEGFR1, and VEGFR2 were measured with Western blot. RESULTS: The immunostain of alpha-SMA were positive in HK-2 cell induced by TGFbeta1 at the concentration of 5 and 8 ng/ml for 72 h. The expression of alpha-SMA mRNA was induced by TGFbeta1 in concentration- and time-dependent manners. The expressions of mRNA and protein of VEGF were upregulated by TGFbeta1 at the concentration of 0.1 and 1 ng/ml for 72 h and at the concentration of 8 ng/ml for 12 h and 24 h when compared with the control. But expressions of mRNA and protein of VEGF were downregulated by TGFbeta1 at the concentration of 3, 5, and 8 ng/ml for 72 h and at the concentration of 8 ng/ml for 36, 48, and 72 h, respectively. Meanwhile, Protein levels of VEGFR1 and VEGFR2 were upregulated by TGFbeta1 in concentration- and time- dependent manners. CONCLUSIONS: Increased expression of VEGFR1 and VEGFR2 and two-phase change in VEGF expression occurred in the process of tubular epithelial transdifferentiation induced by TGFbeta1. Reduced expression of VEGF may contribute to tubular epithelial transdifferentiation in a vicious circle.

[Effect of BMP-7 on the transdifferentiation of cultured human tubular epithelial cell induced by TGF-beta1].

OBJECTIVE: To observe the effect of bone morphogenetic protein-7 (BMP-7) on the transdifferentiation of cultured human tubular epithelial cell (HKC) induced by TGF-beta1 and to elucidate its possible mechanism. METHODS: The cultured HKC cells were divided into 5 groups: serum-free group (negative control); single TGF-beta1 treated group (positive control); single BMP-7 treated group; combined TGF-beta1 and BMP-7 treated group; and BMP-7 pre-treated group. Expression of keratin of HKC cells was assessed by indirect enzyme immunohistochemistry (IEI), expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin by immunohistological method, percentage of alpha-SMA positive HKC cells by flow cytometry, and mRNA expression of alpha-SMA, TGF-beta1, and TGF-beta type II receptor by reverse transcription PCR. RESULTS: The expression of alpha-SMA and the percentage of alpha-SMA positive HKC cells markedly increased after having been treated by TGF-beta1 while the expression of E-cadherin and keratin decreased. In the group pre-treated with BMP-7 (50 ng/ml) and then added with TGF-beta1 (8 ng/ml), expression of alpha-SMA was significantly lower than in the positive control group, while expression of E-cadherin and keratin significantly higher than in the positive control group. Measurement of the percentage of alpha-SMA positive HKC found significant deference between the combined TGF-beta1 and BMP-7 treated group and the positive control group (9.7% vs 19.8%; 5.8% vs 19.8%; P < 0.05). Significant difference existed between the BMP-7 (50 ng/ml) pre-treated group and the positive control group (8.7% vs 19.8%, P < 0.05). mRNA expression of alpha-SMA was measured by RT-PCR and the results showed that it significantly decreased in the group treated or pre-treated with BMP-7 (50 ng/ml) (15% and 12% of the results in the positive control group, respectively). The mRNA expression levels of both TGF-beta1 and its type II receptor significantly decreased (28% and 19%; 47% and 36%, compared with the positive control group, respectively). CONCLUSION: Transdifferentiation of cultured renal epithelial cell induced by TGF-beta1 can be inhibittd by certain levels of BMP-7, cultured together with TGF-beta1 or pretreated. BMP-7 can prevent and inhibit the mRNA expression of TGF-beta1 and its type II receptor, which may be an important mechanism by which BMP-7 inhibit the transdifferentiation of renal tubular epithelial cell.