Clinical manifestations and risk factors of shock in children with multisystem inflammatory syndrome.

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) is a novel disease associated with COVID-19. The COVID-19 epidemic peaked in May 2022 in Taiwan, and we encountered our first case of MIS-C in late May 2022. We aimed to present patients' clinical manifestations and identify risk factors for shock. METHODS: We included patients diagnosed with MIS-C at two medical centers from May 2022 to August 2022. We separated those patients into two groups according to whether they experienced shock. We collected demographic, clinical manifestation, and laboratory data of the patients and performed statistical analysis between the two groups. RESULTS: We enrolled 28 patients, including 13 (46 %) with shock and 15 (54 %) without shock. The median age was 6.4 years (IQR: 1.9-7.5). In single variable analysis, patients with shock tended to be older, had more neurological symptoms, more conjunctivitis and strawberry tongue, lower lymphocyte count, lower platelet counts, and higher C-reactive protein, higher procalcitonin, higher ferritin, and higher D-dimer levels than those without shock. The area under the ROC curve that used procalcitonin to be the risk factor of shock with MIS-C was 0.815 (95 % CI 0.644 to 0.987). The cutoff value obtained by ROC analysis of procalcitonin was 1.68 ng/mL. With this cutoff, the test characteristics of procalcitonin were as follows: sensitivity 77 %, specificity 93 %, positive predictive value 91 %, negative predictive value 82 %. Multivariable analysis revealed that procalcitonin was the only independent risk factor of shock with MIS-C on admission (OR, 26.00, 95 % CI, 1.01-668.89). CONCLUSIONS: MIS-C patients with high initial procalcitonin levels have higher risks of experiencing shock and may need ICU admission.

[Role of serum neutrophil elastase determination in the diagnosis of acute exacerbation of asthma in preschool children].

OBJECTIVE: To study the role of serum neutrophil elastase (NE) level in acute exacerbation of asthma in preschool children. METHODS: A total of 85 preschool children who were diagnosed with asthma between January 2008 and January 2010 were classified into acute exacerbation group (n=44) and non-acute exacerbation group (n=41). Thirty-five children who received physical examination served as the control group. The enzyme-linked immunosorbent assay was used to determine the serum levels of NE and interleukin-8 (IL-8). The receiver operating characteristic (ROC) curve was used for NE evaluation. RESULTS: Both the acute and non-acute exacerbation groups had higher serum levels of NE and IL-8 than the control group, and the acute exacerbation group had significantly higher serum levels of NE and IL-8 than the non-acute exacerbation group (P<0.05). The serum level of NE was positively correlated with that of IL-8 (r=0.48, P<0.05). With serum NE level >27.73 µg/L as the cut-off value for diagnosing acute exacerbation of asthma, the sensitivity was 65.9%, the specificity was 95.1%, and the area under the ROC curve was 0.87 (P<0.01). CONCLUSIONS: The determination of serum NE level in preschool children with asthma helps to diagnose the acute exacerbation of asthma.

Clinical characteristics and factors associated with severe COVID-19 in hospitalized children during the SARS-CoV-2 Omicron pandemic in Taiwan.

BACKGROUND: Since April 2022, a notable increase in COVID-19 cases with the rapid spread of the SARS-CoV-2 Omicron variant has been reported in Taiwan. In the epidemic, children were one of the most vulnerable groups, so we analyzed their clinical presentations and factors associated with severe complications of COVID-19 in children. METHODS: We included hospitalized patients under 18 years old with lab-confirmed SARS-CoV-2 infection from March 1, 2022, to July 31, 2022. We collected the demographic and clinical characteristics of the patients. Patients requiring intensive care were defined as severe cases. RESULTS: Among the 339 enrolled patients, the median age was 31 months (interquartile range (IQR), 8-79.0 months); and 96 patients (28.3%) had underlying diseases. Fever was noted in 319 patients (94.1%) with a median duration of two days (IQR 2-3 days). Twenty-two patients (6.5%) were severe cases, including 10 patients (2.9%) with encephalopathy with abnormal neuroimaging and ten patients (2.9%) with shock. Two patients (0.6%) died. Patients with congenital cardiovascular disease (aOR: 21.689), duration of fever up to four days or more (aOR: 6.466), desaturation (aOR: 16.081), seizure (aOR: 20.92), and procalcitonin >0.5 ng/mL (aOR: 7.886) had a higher risk of severe COVID-19. CONCLUSIONS: Vital signs need close monitoring, early management and/or intensive care may be applied in COVID-19 patients with congenital cardiovascular diseases, fever lasting ≥4 days, seizures, desaturation and/or elevated procalcition since they are at higher risks of severe diseases.

[Risk factors of melamine-contaminated milk powder related urolithiasis: a multicenter nested case-control study].

OBJECTIVE: To further confirm and clarify the risk factors of melamine associated urolithiasis. METHODS: Case control research was performed in 6 centers from 5 provinces/cities in China. Children less than 36 months old were screened for urolithiasis and recruited in the study. The children with urolithiasis were included as cases and those without urolithiasis as controls. The children with congenital abnormality of urinary tract were excluded. According to the case:control ratios of 1:1, we sampled the controls from healthy children screened randomly. Due to the complete missing data on factors of vomiting/diarrhea/fever in control group of Center 4, we analyzed the data from 6 centers and 5 centers respectively. The possible influencing factors for urolithiasis including melamine concentration, birth type, age, feeding style and history of vomiting or diarrhea or fever were analyzed by Logistic analysis. RESULTS: There were 1 329 cases and 1 317 controls with a mean age of 18.4 months. The analysis of data from 6 centers showed the children fed with high melamine formula were 6.26 times more likely to have stones (P<0.01) than those with non melamine formula. Preterm infants were 2.03 times (P<0.01) more likely to have urolithiasis than term infants. The children aged less than 0.5 year, 0.5 to 1 year, 1 to 2 year, 2 to 2.4 year were 2.78 (P<0.01), 2.61 (P<0.01), 2.09 (P<0.01), 1.57 (P<0.01), 1.44 (P<0.05) times more likely to have stones than those more than 2.5 year. Boys were 1.19 times more likely to have stones than girls. Children fed with formula alone were 1.94 times (P<0.01) more likely to have stones than those with formula and breast milk. The analysis of data from 5 centers showed that children fed with high melamine formula were 4.38 times (P<0.01) more likely to have stones compared with those with non melamine formula. Children aged less than 1 year and 1 to 1.9 year were 2.24 (P<0.01) and 1.31 (P<0.05) times more likely to have stones than those more than 2 year. The children fed with formula alone were 1.67 times (P<0.01) more likely to have stones compared to those with formula and breast milk. The children with any two symptoms of vomiting, diarrhea and fever were 15.21 times (P<0.05) more likely to have urolithiasis. The multiple logistic regression model confirmed that above risk factors were independent risk factors for urolithiasis. CONCLUSION: We confirm that the children fed with high melamine infant formula, preterm infant, boy, children fed with formula alone, and the children with symptoms of vomiting or diarrhea or fever are more likely to have urolithiasis. We also found the risk for urolithiasis decreased with age.

[Clinical and pathological features of Alport syndrome in children].

OBJECTIVE: To study the clinical and pathological features of Alport syndrome in children. METHODS: The clinical and histopathological data of 10 hospitalized children with Alport syndrome from February 2007 to February 2009 were retrospectively reviewed. RESULTS: There were 7 males and 3 females, with the age ranging from 2 years to 6 years and 7 months (mean 3 years and 2 months). Five of 10 cases had positive family history. X-linked dominant inheritance Alport syndrome was diagnosed in 8 cases, and autosomal recessive inheritance Alport syndrome in 2 cases. Recurrent gross hematuria was found in 5 cases, hematuria and proteinuria in 3 cases, massive proteinuria in 1 case, and nephritic syndrome in 1 case. Under the light microscope, 8 cases presented with mesangial proliferation glomerulonephritis, and 2 cases with focal segmental glomerulosclerosis. Immunofluorescence assay showed that all cases had IgM deposition in glomerulus. Only 1 case showed typical glomerular basement membrane (GBM) pathological changes. All cases showed abnormal alpha-chain distribution in renal collagen IV. CONCLUSIONS: The children with Alport syndrome have diverse clinical manifestations. Characteristic histopathological presentations could not be found under a light microscope, mesangial proliferation glomerulonephritis is the dominant pathological change, and IgM deposition in glomerulus is common. The GBM pathological change in children is not common. Immunofluorescence assay of alpha-chain in collagen IV is needed for the diagnosis of Alport syndrome.

MiR-29b expression is altered in crescent formation of HSPN and accelerates Ang II-induced mesangial cell activation.

BACKGROUND: MicroRNA-29b (miR-29b) has been suggested to possess pro-inflammatory activity, which can partially be explained by the repression of tumor necrosis factor alpha protein three antibody (TNFAIP3). Meanwhile, it also promotes thyroid cell proliferation via Smad signaling pathways. The present study aimed to elucidate the role of miR-29b in Henoch Schönlein purpura nephritis (HSPN) and its underlying molecular mechanism in angiotensin II (Ang II)-induced human glomerular mesangial cell (HGMC) activation. METHODS: We evaluated miR-29b expression in 35 HSPN renal tissues based on crescent formation, glomerular sclerosis, interstitial fibrosis, thrombosis formation and capillary loop necrosis. Meanwhile, HGMCs were cultured, treated with Ang II and then transfected with LV-hsa-miR-29b-1 to induce miR-29b overexpression or LV-hsa-miR-29b-3p-inhibition to inhibit miR-29b expression. Finally, we examined the effects of miR-29b on cell proliferation and release of inflammatory mediators. RESULTS: We observed that miR-29b expression was significantly higher in the crescent group than in the no crescent group. MiR-29b overexpression induced the release of intercellular adhesion molecule-1, interleukin-1ß (IL-1ß), IL-6, IL-8, the increase of CyclinA2, CyclinD1, and cell proliferation. It also could inhibit the expressions of TNFAIP3 and NF-kappa-B-repressing factor (NKRF). Correspondingly, miR-29b inhibition produced the opposite effects and increased the expression of TNFAIP3 and NKRF. CONCLUSION: MiR-29b expression is altered in crescent formation of HSPN and accelerates Ang II-induced mesangial cell proliferation and release of inflammatory mediators.

[NADPH oxidase-derived reactive oxygen species involved in angiotensin II-induced monocyte chemoattractant protein-1 expression in mesangial cells].

OBJECTIVE: To investigate the origin of oxidative stress induced by angiotensin II (AngII) in human mesangial cells and the role of reactive oxygen species (ROS) in AngII-induced monocyte chemoattractant protein-1 (MCP-1) expression. METHODS: MCP-1 expression was determined by real time RT-PCR. ROS production was measured by DCFDA fluorescence. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was examined by lucigenin chemiluminescence. p47phox and p67phox translocation was assayed by Western blot. Twenty-four male mice were randomly divided into three groups: the control, the AngIIinfusion [AngII 400 ng/(kg.min)], and the apocynin treatment. AngII was infused by subcutaneously osmotic minipump for 14 days. Urinary albumin and 8-isoprostane excretion were measured by ELISA. RESULTS: In cultured human mesangial cells, AngII induced the MCP-1 expression in a dose-dependent manner with 3.56 fold increase as compared with the control. AngII increased intracellular ROS production as early as 3 min with the peak at 60 min and was in a time and dose-dependent. Incubation with different dosages of AngII (1 nmol/L, 10 nmol/L, and 100 nmol/L AngII) for 60 min, ROS production increased at 1.82, 2.92, and 4.08 folds respectively. AngII-induced ROS generation was sensitive to diphenyleneiodonium sulfate (DPI, 10 micromol/L) and apocynin (500 micromol/L), two structurally distinct NADPH oxidase inhibitors. In contrast, inhibitors of other oxidant-producing enzymes, including the mitochondrial complex Iinhibitor rotenone, the xanthine oxidase inhibitor allopurinol, the cyclooxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguiaretic acid, the cytochrome P450 oxygenase inhibitor ketoconazole and the nitric oxide synthase inhibitor G-nitro-L-arginine methyl ester were without an effect. AngII-induced ROS generation was inhibited by the AT1 antagonist losartan (10 micromol/L) but not the AT2 antagonist PD123319 (10 micromol/L). AngII treatment induced translocation of cytosolic of p47phox and p67phox to the membrane. The antioxidants almost abolished AngII-induced MCP-1 expression. AngII infusion increased urinary and p67 translocation by 2.69-, 2.97-, and 2.67-fold, respectively. CONCLUSIONS: NADPH oxidase-derived ROS is involved in AngII-induced MCP-1 expression. Inhibition of NADPH oxidase alleviates AngII-induced renal injury.

Comparative study of urethroplasties to reduce urethral strictures in patients with severe hypospadias.

OBJECTIVE: Transverse island pedicle flap (TIPF) plus transected urethral plate-preserving urethroplasty is increasingly used for treatment of severe hypospadias. We aimed to reduce the occurrence of urethral strictures in patients undergoing such procedures. METHODS: Sixty-five patients with severe hypospadias were enrolled. Thirty-two patients underwent onlay-tube-onlay urethroplasty (Group A), and 33 patients underwent modified Duplay urethroplasty (Group B). Postoperative complications were recorded, including fistulas, urethral strictures, and diverticula. RESULTS: Three patients (9.4%) in Group A and 10 patients (30.3%) in group B had urethrocutaneous fistulas. Three patients (9.4%) in Group A and 0 patients (0%) in Group B had urethral strictures. No patient in the two groups had symptoms of diverticulum or penile chordee. The results of uroflowmetry were better in Group B than Group A, when comparing uroflow patterns. CONCLUSIONS: TIPF plus transected urethral plate-preserving urethroplasty can lower the occurrence of stricture, which is a challenging complication. The occurrence of stricture was lower in patients who underwent modified Duplay urethroplasty, and neourethral function and quality were better in these patient. Thus, this modified procedure can be used for treatment of severe hypospadias.

CREB and Sp1 regulate the human CD2AP gene promoter activity in renal tubular epithelial cells.

The human CD2-associated protein (CD2AP) is involved in several molecular signaling pathways and is an important factor responsible for nephrotic syndrome. Here we report the identification of the transcription start point and promoter region of the human CD2AP gene in renal tubular epithelial cells. With luciferase assays and deletion analysis, we found that the region between -558 and -1bp ahead of the transcription start point is indispensable for the promoter activity of the human CD2AP gene. A CREB site and two Sp1 sites were essential for maintaining the basal transcriptional activity of the human CD2AP promoter. Overexpression of phosphorylated CREB and Sp1 transactivated the human CD2AP promoter, whereas small interfering RNA-mediated blockage of CREB and Sp1 genes expressions inhibited markedly its activity. These findings provide the first analysis of the human CD2AP gene promoter and demonstrate that not only CREB but also Sp1 plays a critical role in regulating basal CD2AP promoter activity in renal tubular epithelial cells.

[Effects of Par-4 gene silencing induced by siRNA on the expression of Smac gene, activity of caspase-3, and apoptosis of human bone marrow mesenchymal stem cells].

OBJECTIVE: To investigate the effects of Par-4 gene silencing induced by siRNA on the expression of Smac gene, activity of caspase-3, and apoptosis of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: Bone marrow was obtained from a healthy young man and hBMSCs were isolated and cultured. Two siRNAs (Par-4-siRNA-1 and -2) targeting Par-4 gene were chemically synthesized. Eukaryocytic expression vectors containing these Par-4 siRNA sequences were established and transfected into the hBMSCs. The hBMSCs were divided into 4 groups: non-transfected hBMSCs (normal control group), blank Pae-4 plasmid transfected hBMSCs (Par4 control group), Par4-siRNA-1 transfected hBMSCs, and Par-4-siRNA-2 transfected hBMSCs. The expression of Par-4 mRNA was detected by real-time PCR. Another hBMSCs were inoculated in DMEM and divided into 4 groups: non-transfected normal hBMSCs, glutamate (an apoptosis inducer) + non-transfected hBMSC group, glutamine + Par-4-siRNA-1 hBMSC group, and glutamate + Par4-SiRNS-2 hBMSC group. Flow cytometry was used to detect the apoptotic rate. The relative activity of caspase-3 was determined by colorimetric assay. Western blotting was used to detect the Smac protein expression. RESULTS: The relative mRNA expression levels of Par-4 gene of the Par-4-siRNA-1 hBMSCs, Par-4 SiENA-2 hBMSCs, and Par-4 control hBMSCs were 0.12 +/- 0.03, 0.33 +/- 0.09, and 0.97 +/- 0.02 respectively, decreased by 88%, 67%, and 3% respectively compared with that of the normal control. The percentages of apoptotic cells of the glutamate + Par-4-siRNA-1 hBMSCs was (37.2 +/- 6.3)%, significantly lower than that of the glutamate + non-transfected hBMSC group [(58.9 +/- 8. 9)%, F = 58.26, P < 0.01). The Smac protein expression level of the glutamate + non-transfected hBMSC group was significantly higher than that of the normal control group (P < 0.01); however, the Smac protein expression level of the Par-4-siRNA-1 hBMSC group was significantly lower than that of the glutamate + non-transfected hBMSC group (P < 0.01). The caspase-3 activity of the glutamate + Par-4-siRNA-1 hBMSC group was significantly lower than that of the glutamate + non-transfected BMSC group (P < 0.01). CONCLUSION: Par-4-siRNA-1 inhibits markedly the apoptosis of the hBMSCs induced by glutamate. Par-4 gene silencing induced by siRNA inhibits the apoptosis of hBMSCs. The mechanism of the inhibition may be closely related to suppression of the up-regulation of Smac gene expression and caspase-3 activity.

Use of off-label nephrology-related drugs in hospitalized pediatric patients: a retrospective study.

BACKGROUND: The information about the use of off-label drugs in pediatric nephrology is still lacking, which leads to increased adverse reactions and medical disputes. We retrospectively analyzed the use of off-label drugs in the in-patient ward of the nephrology department of Nanjing Children's Hospital, China in order to provide more complete information about the use of drugs for children. METHODS: Proportional stratified random sampling was applied to select patients with renal diseases aged 1 month to 18 years, who were admitted to the hospital from October 1, 2012 to September 30, 2013. All nephrology-related drugs prescribed in the hospitalization period and take-home drugs prescribed on discharge were recorded and evaluated as off-label drugs or not from three different perspectives: person-time, prescription, and drug category. RESULTS: From 385 person-times of patients with 1424 prescriptions, according to the ratio between off-label drugs and person-times, drug prescriptions, and drug products, the rates of off-label drugs were 40.78%, 16.64%, and 31.43%, respectively. Low-molecular-weight heparin, alfacalcidol and diltiazem were the most commonly used off-label drugs. Infants and younger children were the high-risk population of off-label drug use. The high rate off-label nephrology-related drug use in children was mainly related to lacking clinical research into drugs in children and the pace of drug label's revision, which cannot follow the development of medical science. CONCLUSION: Approximaely half of pediatric patients with renal diseases are usually prescribed with off-label nephrology-related drugs. Analyzing the off-label conditions from different perspectives may lead to various results. More clinical research into drugs for infants and younger children is needed so as to update drug descriptions.

Heat shock protein 70-2 and tumor necrosis factor-α gene polymorphisms in Chinese children with Henoch-Schönlein purpura.

BACKGROUND: Henoch-Schönlein purpura (HSP) or IgA-associated vasculitis is related to immune disturbances. Polymorphisms of the heat shock protein 70-2 gene (HSP70-2) and the tumor necrosis factor-a gene (TNF-α) are known to be associated with immune diseases. The purpose of this study was to investigate the likely association of HSP70-2 (+1267A/G) and TNF-α (+308A/G) gene polymorphisms with HSP in children. METHODS: The polymerase chain reaction restriction fragment length polymorphism method was used to detect the HSP70-2 and TNF-α polymorphisms in 205 cases of children with HSP and 53 controls; and the association of these polymorphisms with HSP and HSP nephritis (HSPN) was analyzed. RESULTS: The G/G genotypic frequencies at the +1267A/G position of HSP70-2 in the HSP group (22.9%) were significantly higher than those in the healthy control group (9.4%) (χ(2)=4.764, P<0.05). The frequencies of the A/A, A/G and G/G genotypes of HSP70-2 in patients in the nephritis-free group and the HSPN group showed no statistically significant difference. The A/A genotype frequency at the +308G/A position of TNF-α in the HSP group was 8.3%, which was higher than that in the control group (χ(2)=6.447, P<0.05). The A allele frequency of TNF-α in the HSP group was higher than that in the control group, with a statistically significant difference (χ(2)=7.241, P<0.05). CONCLUSIONS: The HSP70-2 (+1267A/G) and TNF-α (+308G/A) gene polymorphisms were associated with HSP in children. The G/G homozygosity of HSP70-2 and the A/A homozygosity of TNF-α may be genetic predisposing factors for HSP.

[Inhibition effects of par-4 antisense oligodeoxynucleotide on apoptosis of PC12 cell induced by glutamate is mediated by ERK1/2].

OBJECTIVE: To investigate the inhibition effects of par-4 antisense oligodeoxynucleotide on apoptosis of PC12 cell induced by glutamate and its signal transduction mechanism. METHODS: (1) Cationic lipid-mediated par-4 antisense oligodeoxynucleotide (Par-4-AS-ODN) was transfected into PC12 cells before they were treated with glutamate. Mismatch oligodeoxynucleotide (MS-ODN) were also transfected into cells as controls. (2) Morphological observation and the detection of anti-apoptosis effects of par-4-AS-ODN on PC12 cells were done with the Laser Scanning confocal Microscope by double staining the cells with acridine orange/ethidium bromide (AO/EB), addition to with flow cytometry. (3) Western blot was used to detect the protein levels of par-4 and phosphorylated ERK(1/2) at threonine-202 and Tyrosine-204. RESULTS: (1) Par-4-AS-ODN significantly suppressed up-regulation of the par-4 protein levels induced by glutamate in PC12 cells. (2) Par-4-AS-ODN could resist the decrease of phosphorylated ERK(1/2) (Thr202/Tyr204) induced by glutamate in PC12 cells. (3) Par-4 AS-ODN could inhibit apoptosis of PC12 cells induced by glutamate. But its inhibition effect could be eliminated by PD98059, a selective MEK(1) inhibitor which could inhibit phosphorylation of ERK(1/2). CONCLUSION: Par-4 AS-ODN may inhibit apoptosis of PC12 cells induced by glutamate, and its inhibition effects may be medicated by the activation of ERK(1/2).

[Suppressive effects of par-4 antisense oligodeoxynucleotide on up-regulation of intracellular calcium concentration in PC12 cell induced by glutamate and its anti-apoptosis effects].

OBJECTIVE: To investigate the uppressive effects of par-4 antisense oligodeoxynucleotide on the up-regulation of intracellular calcium concentration in PC12 cell induced by glutamate and its anti-apoptosis effects. METHODS: Cationic lipid-mediated par-4 antisense oligodeoxynucleotide (par-4-AS-ODN) was transfected into PC12 cells and followed by glutamate for treatment. Mismatch oligodeoxy-nucleotide (MS-ODN) was used as the control. Morphological assessment and evaluation of the anti-apoptosis effects of par-4-AS-ODN on PC12 cells were performed by laser scanning confocal microscopy by double staining of the cells with Hoechest 33258/propidium iodide (Hoe/PI) and flow cytometry respectively. The mRNA and protein levels of calpain 10 and par-4 were measured by RT-PCR and Western blot. Intracellular calcium concentration was determined by using laser scanning confocal microscope with Fura-2/AM as the fluorescent dye. RESULTS: Par-4-AS-ODN repress the increase of par-4 protein in PC12 cell (52.3 +/- 5.0 vs 90.0 +/- 3.2, < 0.01). Par-4-AS-ODN significantly inhibited the apoptosis of PC12 cells induced by glutamate (53% vs 31%, < 0.01). Par-4-AS-ODN significantly suppress the up-regulation of intracellular calcium concentration in PC12 cells induced by glutamate (Rate of fluorescent density: 167.9 +/- 32.4 vs 228.8 +/- 36.8, < 0.01). Par-4-AS-ODN inhibited the increase of calpain 10 mRNA in PC12 cells induced by glutamate (46.3 +/- 3.7 vs 34.8 +/- 2.1, < 0.01). CONCLUSIONS: par-4-AS-ODN enables to inhibit apoptosis of PC12 cells induced by glutamate. The mechanism of the inhibition may be closely related to suppression of the up-regulation of intracellular calcium concentration and calpain transcription expression.

[Expression of monocyte chemoattractant protein-1 in experimental rat glomerulonephritis is mediated by NF-kappaB/IkappaB signal pathway].

OBJECTIVE: To investigate the role of NF-kappaB/IkappaB signal pathway in mediating the expression of monocyte chemoattractant protein-1 (MCP-1) in experimental rat glomerulonephritis. METHODS: Nephrotoxic serum nephritis (NTN) was induced by injection of anti-GBM antibody into the tail veins of rats. Electrophoretic mobility shift assay (EMSA) and Western Blot were used to detect the activation of NF-kappaB, nuclear translocation of p65 subunit and degradation of IkappaBalpha and IkappaBbeta in rat renal tissue. MCP-1 expression in glomeruli and renal tubules was also assessed by immunohistochemistry and ribonuclease protection assay. This was further correlated with the activation of NF-kappaB. RESULTS: There was an obvious expression of MCP-1 in glomeruli and renal tubules. Significant up-regulation of NF-kappaB activation, nuclear translocation of p65 subunit, and degradation of IkappaBalpha and IkappaBbeta were also observed in NTN rat renal tissue, as compared to the control group. A positive correlation was noted between NF-kappaB activation and MCP-1 expression. CONCLUSIONS: NF-kappaB/IkappaB signal pathway may play an important pathogenetic role in glomerulonephritis, with mediating the expression of MCP-1.

[Induction of monocyte chemoattractant protein-1 expression in human mesangial cells by angiotensin II: role of c-Jun N-terminal kinase-c-Jun/activator protein-1 signal pathway].

OBJECTIVE: To investigate the role of c-Jun N-terminal kinase (JNK)-c-Jun/activator protein-1 (AP-1) signal pathway in expression of monocyte chemoattractant protein-1 (MCP-1) in experimental rat glomerulonephritis. METHODS: Nephrotoxic sera nephritis (NTN) was induced by injection of anti-GBM antibody into the tail veins of rats. Electrophoretic mobility shift assay (EMSA) and non-radioactive kinase assay were used to detect the activity of AP-1 and JNK in kidneys and angiotensin II-stimulated human mesangial cells. Ribonuclear protection assay was used to detect MCP-1 expression in cultured human mesangial cells. RESULTS: Significant up-regulation of JNK and AP-1 was observed in NTN rats (3.82 +/- 0.58) folds and (5.36 +/- 0.61) folds, as compared with the controls. Supershift assay demonstrated that c-Jun and c-Fos were the predominant subunits involved. Activation of JNK and AP-1 significantly correlated with MCP-1 expression in NTN rats. Angiotensin II enhanced the expression of MCP-1 and activation of JNK and AP-1 in cultured human mesangial cells in a dose-dependent manner, with maximal stimulation seen at 100 nmol/L (20.99 +/- 4.71) folds, (6.91 +/- 1.65) folds and (7.82 +/- 1.32) folds respectively. Significant down-regulation of AP-1 activation and MCP-1 expression were observed in angiotensin II-induced human mesangial cells pretreated with JNK specific inhibitor SP600125. CONCLUSIONS: Angiotensin II and MCP-1 may play an important role in glomerulosclerosis via the JNK-c-Jun/AP-1 signal pathway.

[Effect of curcumin on extracellular matrix accumulation in the glomeruli in nephrotoxic sera nephritis rats].

OBJECTIVE: To observe whether curcumin could inhibit the accumulation of the collagen IV and fibronectin in the glomeruli in nephrotoxi sera nephritis rats. METHODS: Seventy-two healthy male Sprague-Dawley rats were divided into three groups, with 24 animals in each group. For normal control group, normal saline (0.5 ml/d) was injected through intra-caudal-vein for two days, and at the same time normal saline (0.5 ml/kg) was also daily administered intraperitoneally. For nephrotoxic sera nephritis group, nephrotoxic sera (0.5 ml/d) was injected through the tail vein for two days and dimethyl sulfoxide (0.5 ml/kg) was given intraperitoneally daily. For curcumin group, nephrotoxic sera was injected as above and meanwhile curcumin (50 mg.kg(-1).d(-1)) was administered intraperitoneally every day. Six rats in each group were killed on the 3rd, 7th, 14th and 28th day. Their renal tissue was fixed in 10% formalin for examining the expression of collagen IV and fibronectin. RESULTS: Minimal staining of collagen IV and fibronectin was detected in the basement membrane of normal control rats glomeruli. In the nephrotoxic sera nephritis rats and curcumin treated nephrotoxic sera nephritis rats, the accumulation of collagen IV and fibronectin was increased progressively, with significant difference in the accumulation of collagen IV (P<0.01) between these two groups at the same time points, while the significant difference in fibronectin accumulation (P<0.05) appeared only after the 7th days. CONCLUSION: Curcumin can reduce the accumulation of collagen IV and fibronectin in the glomeruli. Hence we postulated that curcumin might have beneficial effect for retarding glomerulosclerosis.

[Mechanisms of decorin inhibiting epithelial-to-mesenchymal transition induced by transforming growth factor beta1 in renal tubular epithelial cells].

OBJECTIVE: To investigate the mechanisms of decorin inhibiting epithelial-to-mesenchymal transition (EMT) induced by transforming growth factor beta1 (TGF-beta1) in renal tubular epithelial cells. METHOD: HK-2 cells in vitro were divided into 4 groups: (1) negative control group; (2) decorin group, added with decorin 100 ng/ml ; (3) TGF-beta1 group, added with TGF-beta1 10 ng/ml; (4) decorin and TGF-beta1 group, added with decorin 100 ng/ml and TGF-beta1 10 ng/ml. The protein level of phosphor-ERK, phosphor-PI3K, phosphor-Smad(3) and beta-catenin was detected by Western blotting method. The snail mRNA level was tested by real time-PCR, while the lymphoid enhancer factor-1 (LEF-1) mRNA level was measured by RT-PCR. RESULTS: The snail (2.59 +/- 0.70:1.02 +/- 0.13) and LEF-1 mRNA (1.85 +/- 0.08:0.30 +/- 0.11) were significantly up-regulated, meanwhile the protein level of phosphor-ERK (1.11 +/- 0.09:0.47 +/- 0.07), phosphor-PI3K (14.79 +/- 1.02:2.48 +/- 0.06), phosphor-Smad(3) (0.95 +/- 0.02:0.08 +/- 0.01) and beta-catenin (1.46 +/- 0.20:0.49 +/- 0.05) were significantly increased in TGF-beta1 group compared to control group, while there were no statistically significant difference in all figures between control group and decorin group. The phosphor-ERK protein level (0.58 +/- 0.08) and the snail mRNA level (1.24 +/- 0.03) were significantly down-regulated in TGF-beta1 and decorin group compared to TGF-beta1 group, however there were no statistically significant differences in the level of phosphor-PI3K (15.84 +/- 1.64), phosphor-Smad(3) (0.90 +/- 0.04) and beta-catenin (1.42 +/- 0.09) between these two groups. CONCLUSION: Decorin inhibited EMT induced by TGF-beta1 which may be through blocking the ERK signal transduction pathway.

SP600125, an inhibitor of c-Jun NH2-terminal kinase, blocks expression of angiotensin II-induced monocyte chemoattractant protein-1 in human mesangial cells.

BACKGROUND: We investigated the role of c-Jun NH2-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, in the expression of angiotensin II (Ang II)-induced monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-1 (TGF-1), and in the production of fibronectin (FN), by human mesangial cells (HMCs). METHODS: JNK activation in cultured human mesangial cells was determined by Western blotting with an antibody against the phosphorylated Ser63 residue of c-Jun. Binding of the activator protein (AP-1) to the MCP-1 AP-1 motif was detected via the electrophoretic mobility shift assay (EMSA). The transient luciferase reporter was used to examine MCP-1 promoter activity; an RNase protection assay and ELISA were used respectively to detect the expression of MCP-1 mRNA and production of MCP-1, TGF-beta and FN. RESULTS: Anthra (1,9-cd) pyrazol-6(2H)-one (SP600125), a pharmacological inhibitor of JNK, almost completely abolished Ang II-induced Ser63 phosphorylation of c-Jun at concentrations of 5-20 micromol/L: JNK activity was reduced by 75% with 10 micromol/L SP600125, and by 90% with 20 micromol/L. Ang II increased AP-1 binding to the MCP-1 AP-1 motif in a time-dependent manner, as detected by EMSA, while SP600125 effectively blocked this increased AP-1 binding in a concentration-dependent manner. Treatment with 100 nmol/L Ang II led to a steady increase in MCP-1 mRNA expression, and to an enhanced production of MCP-1, TGF-beta and FN. These effects were blocked by SP60025 in a dose-dependent manner. SP600125 also reduced MCP-1 mRNA stability: the halflife of MCP-1 mRNA was approximately 5 hours in cells treated with Ang II only, but was reduced to 2 hours when treated with a combination of Ang II and SP600125. CONCLUSIONS: These results show that the JNK/AP-1 pathway is involved in the expression of MCP-1 and TGF-beta, and in extracellular matrix production. JNK is an important therapeutic target for glomerulonephritis and glomerulosclerosis.

[The effects of core proteoglycan on the expressions of I and III collagen in human renal tubular epithelial cell induced by TGFbeta1 in vitro].

AIM: To explore the roles of core proteoglycan and TGFbeta1 on the expressions of I and III collagen in human renal tubular epithelial cell line(HK-2) in vitro. METHODS: Confluent HK-2 cells were exposed to TGFbeta1 and core proteoglycan for up to 48 h. The cells were divided into four groups. Group (1), negative control group; group(2), single 10 microg/L TGFbeta1 treated group; group (3), 10 microg/L TGFbeta1+10 microg/L core proteoglycan group; group (4), 10 microg/L TGFbeta1+100 microg/L core proteoglycan group. Morphologic characterization of HK-2 cells was shown by invertmicroscope; Precise amounts of I and III collagen mRNA were measured by RT-PCR. RESULTS: After 48 h, morphology of (1) group cells had no changes, most cells were normal shape; (2) group cells took great changes, most cells converted into spindle shape, like fibroblast, (3) and (4) groups, spindle shape cells reduced significantly. In contrast to (1) group, the expressions of I collagen in (2) group from mRNA significant increased by 27.86-fold. The expressions of III collagen increased by 21.83-fold. Comparing (3) and (4) groups to(2) group, the expressions of I collagen from mRNA effectively decreased 36.39% and 53.36%. III collagen expressions increased 26.35% and 47.96%èP<0.05érespectively. But, neither (3) group nor (4) group alone could regulate I and III collagen mRNA to normal levels. CONCLUSION: Core proteoglycan can inhibit the expressions of I and III collagen in HK-2 cells induced by TGFbeta1 in vitro. Possibly, suggest core proteoglycan contribute to the regulation of renal fibrosis.