Depression in public officials during the COVID-19 pandemic in Paraguay: a web-based study.

BACKGROUND: According to the World Health Organization, the coronavirus disease 2019 (COVID-19) pandemic has created situations that have a negative effect on people and threaten their mental health. Paraguay announced the Estado de Emergencia Sanitaria (Presidential Decree No. 3456) on March 16, 2020, which was followed by the imposition of a 24-h restriction on movement order on March 21. Self-quarantine at home may have been the most effective method of preventing the spread of infectious diseases; however, with the global pandemic becoming more prolonged and the consequent lengthening of the 24-h self-quarantine period, it is highly probable that both physical and psychological problems will arise. METHODS: In this study, a web-based cross-sectional method was used to analyze the factors influencing COVID-19-induced depressive feelings in Paraguayan public officials. RESULTS: Public officials reported a high level of depressive symptoms with a high level of apprehension in early stage of COVID-19. In addition, this study identified that when the self-quarantine period increased, levels of depressive feelings also increased. Since self-quarantine is characterized by the requirement that individuals endure an undetermined period within a confined area, it may have caused stress and anxiety, as well as the consequent experience of depressive feelings. CONCLUSIONS: Paraguayan government should develop a program for the delivery of mental health care and services to public officials in COVID-19 Pandemic period. Moreover, a program is required for people facing deteriorating mental health due to social isolation and loneliness caused by social distancing during the prolonged period of self-quarantine. Finally, mental health care programs should be organized in a community-focused way by utilizing online systems to enhance the effectiveness of mental health recovery.

Podocyte hypertrophy precedes apoptosis under experimental diabetic conditions.

Podocyte hypertrophy and apoptosis are two hallmarks of diabetic glomeruli, but the sequence in which these processes occur remains a matter of debate. Here we investigated the effects of inhibiting hypertrophy on apoptosis, and vice versa, in both podocytes and glomeruli, under diabetic conditions. Hypertrophy and apoptosis were inhibited using an epidermal growth factor receptor inhibitor (PKI 166) and a pan-caspase inhibitor (zAsp-DCB), respectively. We observed significant increases in the protein expression of p27, p21, phospho-eukaryotic elongation factor 4E-binding protein 1, and phospho-p70 S6 ribosomal protein kinase, in both cultured podocytes exposed to high-glucose (HG) medium, and streptozotocin-induced diabetes mellitus (DM) rat glomeruli. These increases were significantly inhibited by PKI 166, but not by zAsp-DCB. In addition, the amount of protein per cell, the relative cell size, and the glomerular volume were all significantly increased under diabetic conditions, and these changes were also blocked by treatment with PKI 166, but not zAsp-DCB. Increased protein expression of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, together with increased Bax/Bcl-2 ratios, were also observed in HG-stimulated podocytes and DM glomeruli. Treatment with either zAsp-DCB or PKI 166 resulted in a significant attenuation of these effects. Both PKI 166 and zAsp-DCB also inhibited the increase in number of apoptotic cells, as assessed by Hoechst 33342 staining and TUNEL assay. Under diabetic conditions, inhibition of podocyte hypertrophy results in attenuated apoptosis, whereas blocking apoptosis has no effect on podocyte hypertrophy, suggesting that podocyte hypertrophy precedes apoptosis.

Enhanced glycogen synthase kinase-3ß activity mediates podocyte apoptosis under diabetic conditions.

Glycogen synthase kinase-3ß (GSK-3ß) is involved in the pathogenesis of various kidney diseases. This study was undertaken to examine the changes in GSK-3ß activity in podocytes under diabetic conditions and to elucidate the functional role of GSK-3ß in podocyte apoptosis. In vivo, 32 rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with 6-bromoindirubin-3'-oxime (BIO) for 3 months. In vitro, immortalized mouse podocytes were exposed to 5.6 mM glucose or 30 mM glucose (HG) with or without 10 µM BIO. Western blot analysis and TUNEL or Hoechst 33342 staining were performed to identify apoptosis. Urinary albumin excretion was significantly higher in DM rats, and this increase was significantly abrogated in DM rats by BIO treatment. The protein expression of Tyr216-phospho-GSK-3ß was significantly increased in DM glomeruli and in cultured podocytes exposed to HG. Western blot analysis revealed that the protein expression of Bax and active fragments of caspase-3 were significantly increased, whereas phospho-Akt, ß-catenin, and Bcl-2 protein expression were significantly decreased in DM glomeruli and HG-stimulated podocytes. Apoptosis, determined by TUNEL assay and Hoechst 33342 staining, was also significantly increased in podocytes under diabetic conditions. The changes in the expression of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG-stimulated podocytes were significantly ameliorated by BIO. These findings suggest that enhanced GSK-3ß activity within podocytes under diabetic conditions is associated with podocyte loss in diabetic nephropathy.

The monocyte chemoattractant protein-1 (MCP-1)/CCR2 system is involved in peritoneal dialysis-related epithelial-mesenchymal transition of peritoneal mesothelial cells.

Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) has a role in the process of peritoneal fibrosis (PF), a serious complication in peritoneal dialysis (PD) patients. Even though monocyte chemoattractant protein-1 (MCP-1) was demonstrated to directly increase extracellular matrix (ECM) synthesis, the role of the MCP-1/CCR2 system in PD-related EMT and ECM synthesis in cultured human PMCs (HPMCs) and in an animal model of PD has never been elucidated. In vitro, HPMCs were exposed to 5.6 mM glucose (NG), NG+MCP-1 (10 ng/ml) (NG+MCP-1), or 100 mM glucose (HG) with or without CCR2 inhibitor (RS102895) (CCR2i) or a dominant-negative mutant MCP-1-expressing lentivirus (LV-mMCP-1). In vivo, PD catheters were inserted into 60 Sprague-Dawley rats, and saline (Control, C) (N=30) or 4.25% PD solution (PD) (N=30) was infused for 4 weeks. Twenty rats from each group were treated with empty LV or LV-mMCP-1 intraperitoneally. Snail, E-cadherin, α-smooth muscle actin (α-SMA), and fibronectin protein expression in HPMCs and the peritoneum was evaluated by western blot analysis. Compared with NG cells, Snail, α-SMA, and fibronectin expression was significantly increased, while E-cadherin expression was significantly decreased in HPMCs exposed to HG and NG+MCP-1, and these changes were significantly abrogated by CCR2i (P<0.05). In addition, MCP-1-induced EMT was significantly attenuated by anti-TGF-ß1 antibody. In PD rats, Snail and fibronectin expression was significantly increased in the peritoneum, whereas the ratios of E-cadherin/α-SMA protein expression were significantly decreased (P<0.05). The thickness of the peritoneum and the intensity of Masson's trichrome staining in the peritoneum were also significantly higher in PD rats than in C rats (P<0.05). These changes in PD rats were significantly abrogated by LV-mMCP-1. These findings suggest that the MCP-1/CCR2 system is directly involved in PD-related EMT and ECM synthesis and that this is mediated, at least in part, via TGF-ß1.

The MCP-1/CCR2 axis in podocytes is involved in apoptosis induced by diabetic conditions.

Previous studies have demonstrated the importance of monocyte chemoattractant protein-1 (MCP-1) in the pathogenesis of diabetic nephropathy in terms of inflammation, but the direct role of the MCP-1/CCR2 system on podocyte apoptosis under diabetic conditions has never been explored. In vitro, mouse podocytes were exposed to a medium containing 30 mM glucose (HG) with or without CCR2 siRNA or CCR2 inhibitor (RS102895). Podocytes were also treated with MCP-1 or TGF-ß1 with or without anti-TGF-ß1 antibody, CCR2 siRNA, or CCR2 inhibitor. In vivo, 20 db/m and 20 db/db mice were divided into two groups, and ten mice from each group were treated with RS102895. Western blot and Hoechst 33342 or TUNEL staining were performed to identify apoptosis. HG-induced apoptosis and TGF-ß1 levels were significantly abrogated by CCR2 inhibition. In addition, treatment with MCP-1 directly induced apoptosis via CCR2. Moreover, TGF-ß1- and MCP-1-induced apoptosis were significantly ameliorated by the inhibition of CCR2 and anti-TGF-ß1 antibody, respectively. Glomerular expression of cleaved caspase-3 and apoptotic cells within glomeruli were also significantly increased in db/db mice compared to db/m mice, and these increases were significantly attenuated in db/db + RS102895 mice. These results suggest that interactions between the MCP-1/CCR2 system and TGF-ß1 may contribute to podocyte apoptosis under diabetic conditions.

Apoptosis occurs differentially according to glomerular size in diabetic kidney disease.

BACKGROUND: Apoptosis, which is involved in the process of mesangial cell and podocyte loss in diabetic nephropathy, is known to be regulated by protein kinase B/Akt (Akt). A number of studies have therefore investigated the activity of Akt under diabetic conditions, but the results have not been consistent. In this study, we hypothesized that apoptosis may occur differentially and that Akt may be differentially activated according to glomerular size in diabetic kidney disease. METHODS: Fifty male Sprague-Dawley rats were injected intraperitoneally with diluent (C, n = 25) or streptozotocin (DM, n = 25). After 3 months, glomeruli were isolated using sieves with pore sizes of 250, 150, 125 and 75 µm and then classified into large glomeruli (on the 125-µm sieve, LG) and small glomeruli (on the 75-µm sieve, SG) groups. Western blot analyses for phospho-Akt, apoptosis-related molecules (Bax, Bcl-2, active fragments of Caspase-3 and phospho-p53) and cyclin-dependent kinase inhibitors were performed. CONCLUSIONS: The numbers of total cells and podocytes in isolated glomeruli were determined using transmission electron microscopy. Akt phosphorylation was significantly decreased in DM-LG, while it was significantly increased in DM-SG (P < 0.05). The ratio of Bax/Bcl-2 protein expression and active fragments of Caspase-3 and phospho-p53 protein expression were significantly increased in DM-LG compared to DM-SG and C-SG (P < 0.001 and P < 0.01, respectively). In contrast, the expression of p27(Kip1) and p21(Cip1) was significantly increased in DM-SG compared to DM-LG and C-SG (P < 0.05). The numbers of total glomerular cells and podocytes were significantly decreased in DM-LG (P < 0.05). In conclusion, these data show differential expression of Akt activity and apoptosis-related molecules according to glomerular size in diabetic nephropathy, suggesting that apoptosis may be more operative in more hypertrophic glomeruli, resulting in fewer glomerular cells and podocytes in diabetic nephropathy.

Local kallikrein-kinin system is involved in podocyte apoptosis under diabetic conditions.

The kallikrein-kinin system (KKS) serves as the physiologic counterbalance to the renin-angiotensin system. This study was conducted to examine the changes in the expression of KKS components in podocytes under diabetic conditions and to elucidate the functional role of bradykinin (BK) in diabetes-associated podocyte apoptosis. Thirty-two rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with BK infusion for 6 weeks. Immortalized mouse podocytes were cultured in media containing 5.6 mmol/l glucose (NG), NG + 10(-7) mol/l AII (AII), or 30 mmol/l glucose (HG) with or without 10(-8) mol/l BK. Urinary albumin excretion was significantly higher in DM rats, and this increase was ameliorated by BK. Not only kininogen, kallikrein, and BK B1- and B2-receptor expression but also BK levels were significantly decreased in DM glomeruli and in cultured podocytes exposed to HG. The changes in the expressions of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG- and AII-stimulated podocytes were significantly abrogated by BK. The suppressed KSS within podocytes under diabetic condition was associated with podocyte apoptosis, suggesting that BK may be beneficial in preventing podocyte loss in diabetic nephropathy.

Effects of an oral adsorbent on oxidative stress and fibronectin expression in experimental diabetic nephropathy.

BACKGROUND: Previous studies have demonstrated that AST-120 (Kremezin((R))), a well-known oral adsorbent, inhibits the progression of diabetic (DM) and non-DM chronic kidney disease along with a decrease in oxidative stress. This study was undertaken to investigate whether AST-120 could reduce oxidative stress and ameliorate the development of nephropathy in experimental DM rats with normal renal function. METHODS: Rats were injected with diluent (C, n = 16) or 65 mg/kg streptozotocin intraperitoneally (DM, n = 16), and eight rats from each group were treated with chow containing 5% AST-120. After 3 months, plasma advanced oxidation protein products (AOPP) and total malondialdehyde (MDA) levels, 24-h urinary albumin excretion, and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion were determined by ELISA. Glomerular endothelial nitric oxide synthase (eNOS), subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (gp91phox, p47phox and p22phox), and fibronectin (FN) mRNA and protein expressions were determined by real-time PCR and western blot, respectively. In addition, dichlorodihydrofluorescein diacetate (DCF-DA) staining was performed to detect glomerular reactive oxygen species (ROS) production. RESULTS: Compared to the C group, 24-h urinary albumin excretion was significantly higher in the DM group (P < 0.01), and AST-120 treatment significantly reduced albuminuria in DM rats (P < 0.05). Glomerular eNOS, gp91phox, p47phox and FN expression were significantly increased in DM rats compared to C rats, and these increases in DM glomeruli were significantly abrogated by AST-120 treatment (P < 0.05). The increases in plasma AOPP and MDA levels as well as renal oxidative stress in DM rats, assessed by DCF-DA staining and urinary 8-OHdG excretion rates, were also significantly attenuated by AST-120 treatment (P < 0.05). CONCLUSION: In conclusion, the renoprotective effects of AST-120 in DM nephropathy seem to be associated with the amelioration of enhanced oxidative stress and FN expression under diabetic conditions.

Colchicine attenuates inflammatory cell infiltration and extracellular matrix accumulation in diabetic nephropathy.

Recent studies have demonstrated that an inflammatory mechanism contributes to the pathogenesis of diabetic nephropathy (DN). It is also known that colchicine (Col) can prevent various renal injuries via its anti-inflammatory action. However, the effect of colchicine on DN has never been explored. This study was undertaken to elucidate the effect of colchicine on inflammation and extracellular matrix accumulation in DN. In vivo, 64 rats were injected with diluent (C; n = 32) or streptozotocin intraperitoneally (DM, n = 32). Sixteen rats from each group were treated with Col. In vitro, rat mesangial cells and NRK-52E cells were cultured in media with 5.6 mM glucose (NG) or 30 mM glucose (HG) with or without 10(-8) M Col. Monocyte chemotactic protein-1 (MCP-1) mRNA expression was determined by real-time PCR (RT-PCR), and the levels of MCP-1 in renal tissue and culture media were measured by ELISA. RT-PCR and Western blotting were also performed for intercellular adhesion molecule-1 (ICAM-1) and fibronectin (FN) mRNA and protein expression, respectively, and immunohistochemical staining (IHC) for ICAM-1, FN, and ED-1 with renal tissue. Twenty-four-hour urinary albumin excretion at 6 wk and 3 mo were significantly higher in DM compared with C rats (P < 0.05), and colchicine treatment significantly reduced albuminuria in DM rats (P < 0.05). Col significantly inhibited the increase in MCP-1 mRNA expression and protein levels under diabetic conditions both in vivo and in vitro. ICAM-1 and FN expression showed a similar pattern to the expression of MCP-1. IHC revealed that the number of ED-1(+) cells were significantly higher in DM compared with C kidney (P < 0.005), and this increase was significantly attenuated by Col treatment (P < 0.01). In conclusion, Col prevents not only inflammatory cell infiltration via inhibition of enhanced MCP-1 and ICAM-1 expression but also ECM accumulation in DN. These findings provide a new perspective on the renoprotective effects of Col in DN.

Activation of local aldosterone system within podocytes is involved in apoptosis under diabetic conditions.

Previous studies have shown that mineralocorticoid receptor (MCR) blocker reduces proteinuria in diabetic nephropathy (DN), but the role of aldosterone in podocyte injury has never been explored in DN. This study was undertaken to elucidate whether a local aldosterone system existed in podocytes and to examine its role in podocyte apoptosis under diabetic conditions. In vitro, immortalized podocytes were exposed to 5.6 mM glucose (NG), NG + 24.4 mM mannitol, and 30 mM glucose (HG) with or without 10(-7) M spironolactone (SPR). In vivo, 32 Sprague-Dawley rats were injected with diluent (C, n = 16) or streptozotocin intraperitoneally [diabetes mellitus (DM), n = 16], and 8 rats from each group were treated with SPR for 3 mo. Aldosterone synthase (CYP11B2) and MCR mRNA and protein expression were determined by real-time PCR and Western blot, respectively, and aldosterone levels by radioimmunoassay. Western blot for apoptosis-related molecules, Hoechst 33342 staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to determine apoptosis. CYP11B2 and MCR expression were significantly higher in HG-stimulated podocytes and DM glomeruli compared with NG cells and C glomeruli, respectively, along with increased aldosterone levels. Western blot analysis revealed that cleaved caspase-3 and Bax expression was significantly increased, whereas Bcl-2 expression was significantly decreased in HG-stimulated podocytes and in DM glomeruli. Apoptosis determined by Hoechst 33342 staining and TUNEL assay were also significantly increased in podocytes under diabetic conditions. These changes in the expression of apoptosis-related proteins and the increase in apoptotic cells were inhibited by SPR treatment. These findings suggest that a local aldosterone system is activated and is involved in podocyte apoptosis under diabetic conditions.

Induction of heme oxygenase-1 protects against podocyte apoptosis under diabetic conditions.

Heme oxygenase-1 (HO-1) is an anti-oxidant enzyme normally upregulated in response to oxidant injury. Here we determined the role of HO-1 in podocyte apoptosis in glomeruli of streptozotocin-treated rats and in immortalized mouse podocytes cultured in media containing normal or high glucose. HO-1 expression, its activity, the ratio of Bax/Bcl-2 protein, and active caspase-3 fragments were all significantly higher in isolated glomeruli of diabetic rats and in high glucose-treated podocytes. These increases were inhibited by zinc protoporphyrin treatment of the rats or by HO-1 siRNA treatment of the podocytes in culture. The number of apoptotic cells was also significantly increased in the glomeruli of diabetic rats and in high glucose-treated podocytes. Inhibition of HO-1 accentuated the increase in apoptotic cells both in vivo and in vitro. Our findings suggest that HO-1 expression protects against podocyte apoptosis under diabetic conditions.

Obesity and Hypertension among School-going Adolescents in Peru.

BACKGROUND: Adolescent obesity and hypertension are global public health issues. The burden of adolescent obesity and hypertension in Peru is unclear. The aim of this study was to determine the prevalence of obesity and hypertension and their relationship among school-attending adolescents and to assess the need for health-promoting school programs in the study area. METHODS: A cross-sectional school-based survey was conducted in a randomly selected sample of 952 secondary school adolescents from 11 schools in Lima or Callao, Peru, in 2014. Weight, height, and blood pressure (BP) were measured and categorized. Obesity was defined as ≥ 95(th) percentile in body mass index (BMI) for age and sex. Hypertension was defined as average systolic blood pressure and/or diastolic blood pressure ≥95(th) percentile in BP for sex, age, and height. Chi-square test and univariate logistic regressions were used at a 5% significance level to determine the relationship between BMI and BP category. RESULTS: The mean age of subjects was 14.6 years; 46.4% were boys and 53.6% were girls. The prevalence of overweight and obesity was 20.2% and 9.5% overall, 17.4% and 11.1% for boys, and 22.5% and 8.0% for girls, respectively. The prevalence of hypertension was 26.7% overall, 34.8% for boys, and 19.6% for girls. In both sexes, BMI was strongly associated with BP (p < 0.01). CONCLUSION: The prevalence of obesity and hypertension observed in the study area is relatively high. Overweight and obesity are strongly associated with BP status among adolescents. Health-promoting school programs may reduce the burdens of obesity and hypertension among school-going adolescents.

Energy content estimation by collegians for portion standardized foods frequently consumed in Korea.

The purpose of this study is to estimate Korean collegians' knowledge of energy content in the standard portion size of foods frequently consumed in Korea and to investigate the differences in knowledge between gender groups. A total of 600 collegians participated in this study. Participants' knowledge was assessed based on their estimation on the energy content of 30 selected food items with their actual-size photo images. Standard portion size of food was based on 2010 Korean Dietary Reference Intakes, and the percentage of participants who accurately estimated (that is, within 20% of the true value) the energy content of the standard portion size was calculated for each food item. The food for which the most participants provided the accurate estimation was ramyun (instant noodles) (67.7%), followed by cooked rice (57.8%). The proportion of students who overestimated the energy content was highest for vegetables (68.8%) and beverages (68.1%). The proportion of students who underestimated the energy content was highest for grains and starches (42.0%) and fruits (37.1%). Female students were more likely to check energy content of foods that they consumed than male students. From these results, it was concluded that the knowledge on food energy content was poor among collegians, with some gender difference. Therefore, in the future, nutrition education programs should give greater attention to improving knowledge on calorie content and to helping them apply this knowledge in order to develop effective dietary plans.

The effect of statin on epithelial-mesenchymal transition in peritoneal mesothelial cells.

BACKGROUND: Statins have recently been highlighted for their pleiotropic actions distinct from cholesterol-lowering effects. Despite this interest, it is currently unknown whether statin therapy inhibits peritoneal dialysis (PD)-related epithelial-mesenchymal transition (EMT). METHODS: In vitro, human peritoneal mesothelial cells (HPMCs) were exposed to 5.6 mM glucose (NG) or 100 mM glucose (HG) with or without simvastatin (1 µM). In vivo, PD catheters were inserted into 32 Sprague-Dawley rats, and saline (C, n = 16) or 4.25% peritoneal dialysis fluid (PDF) (PD, n = 16) was infused for 4 weeks. Eight rats from each group were treated with 5 mg/kg/day of simvastatin intraperitoneally. Changes in the protein expression of EMT markers such as E-cadherin, α-SMA, Snail, and fibronectin in HPMCs and the peritoneum were evaluated by Western blot analysis and immunofluorescence or immunohistochemical staining. We also explored whether activation of the mevalonate pathway and its downstream small GTPases were involved in dialysis-related peritoneal EMT and could be inhibited by statin treatment. RESULTS: Compared to NG cells, E-cadherin expression was significantly decreased, while α-SMA, Snail, and fibronectin expression were significantly increased in HPMCs exposed to HG, and these changes were abrogated by simvastatin (p<0.05). In addition, the cobblestone-like appearance of normal HPMCs was converted into a fibroblast-like morphology after HG treatment, which was reversed by simvastatin. These EMT-like changes were also observed in HPMCs treated with geranyl-geranyl pyrophosphate (5 µM). HG significantly increased the protein expression of RhoA and Rac1 in the membrane fractions, and these increases were ameliorated by simvastatin (p<0.05). In PD rats, E-cadherin in the peritoneum was significantly decreased, whereas α-SMA, Snail, and fibronectin expression were significantly increased (p<0.05) compared to C rats. The thickness of the mesothelial layer in the peritoneum were also significantly greater in PD rats than in C rats (p<0.05). These changes of the peritoneum in PD rats were significantly attenuated by simvastatin. CONCLUSION: This study demonstrated that PD-related EMT was mediated via the mevalonate pathway, and statin treatment inhibited the EMT changes in HG-treated HPMCs and PDF-stimulated PD rats. These findings suggest that statins may be a promising therapeutic strategy for preservation of peritoneal membrane integrity in long-term PD patients.

FR167653 inhibits fibronectin expression and apoptosis in diabetic glomeruli and in high-glucose-stimulated mesangial cells.

Previous in vitro studies suggest that the p38 MAPK pathway may be involved in the pathogenesis of diabetic nephropathy, but the consequences of the inhibition of the p38 MAPK pathway have not been well elucidated in diabetic (DM) glomeruli. This study was undertaken to investigate the effect of p38 MAPK inhibitor, FR167653, on fibronectin expression and apoptosis in DM glomeruli and in high-glucose-stimulated mesangial cells (MC). In vivo, 32 Sprague-Dawley rats were injected with diluent (control, N = 16) or streptozotocin intraperitoneally (DM, N = 16). Eight rats from each group were treated with FR167653 for 3 mo. In vitro, rat MC were exposed to medium containing 5.6 mM glucose or 30 mM glucose [high glucose (HG)] with or without 10(-6) M FR167653 for 24 h. Fibronectin mRNA and protein expression were determined by real-time PCR and Western blot, respectively. Western blot for apoptosis-related molecules, terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay, and Hoechst 33342 staining were performed to determine apoptosis. FR167653 ameliorated the increases in fibronectin-to-GAPDH mRNA ratio and protein expression in DM glomeruli by 89 and 79% and in HG-stimulated MC by 70 and 91%, respectively (P < 0.05). Under diabetic conditions, Bcl-2 protein expression was decreased, whereas cleaved caspase-3 protein expression was increased (P < 0.05), and these changes were inhibited by FR167653 treatment. Apoptotic cells were also significantly increased in DM glomeruli and in HG-stimulated MC (P < 0.05), and FR167653 ameliorated these increases in apoptotic cells, both in vivo and in vitro. In conclusion, these findings suggest that the inhibition of the p38 MAPK pathway has a beneficial effect on the development of diabetic nephropathy by inhibiting the increase in fibronectin expression and apoptosis.

MCP-1/CCR2 system is involved in high glucose-induced fibronectin and type IV collagen expression in cultured mesangial cells.

Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that plays an important role in the recruitment of macrophages. Although previous studies have demonstrated the importance of MCP-1 in the pathogenesis of diabetic nephropathy (DN) in terms of inflammation, the role of MCP-1 and its receptor (C-C chemokine receptor 2; CCR2) in extracellular matrix (ECM) accumulation under diabetic conditions has been largely unexplored. This study was undertaken to investigate the functional role of the MCP-1/CCR2 system in high glucose-induced ECM (fibronectin and type IV collagen) protein expression in cultured mesangial cells (MCs). Mouse MCs were exposed to medium containing 5.6 mM glucose (NG), NG+24.4 mM mannitol (NG+M), or 30 mM glucose (HG) with or without mutant MCP-1 (mMCP-1), CCR2 small interfering (si)RNA, or CCR2 inhibitor (RS102895). To examine the relationship between MCP-1 and transforming growth factor (TGF)-beta1, MCs were also treated with TGF-beta1 (2 ng/ml) with or without mMCP-1 or CCR2 siRNA. Transient transfection was performed with Lipofectamine 2000 for 24 h. Cell viability was determined by an MTT assay, mouse and human MCP-1 and TGF-beta1 levels by ELISA, and CCR2 and ECM protein expression by Western blotting. Transfections of mMCP-1 and CCR2 siRNA increased human MCP-1 levels and inhibited CCR2 expression, respectively. HG-induced ECM protein expression and TGF-beta1 levels were significantly attenuated by mMCP-1, CCR2 siRNA, and RS102895 (P < 0.05). MCP-1 directly increased ECM protein expression, and this increase was inhibited by an anti-TGF-beta1 antibody. In addition, TGF-beta1-induced ECM protein expression was significantly abrogated by the inhibition of the MCP-1/CCR2 system (P < 0.05). These results suggest that an interaction between the MCP-1/CCR2 system and TGF-beta1 may contribute to ECM accumulation in DN.