A Forgotten Double-J Ureteric Stent as the Core of a Bladder Stone: A Case Report and Literature Review.

BACKGROUND Double-J (D-J) ureteric stents are widely applied in urological operations as they play a vital role in maintaining postoperative functionality of the patient's urinary system and thereby accelerating recovery. D-J stent encrustation may occur due to prolonged retention and lead to secondary complications. We report the case of a forgotten D-J stent that gradually formed into a bladder stone. CASE REPORT A 54-year-old man was referred to the Urology Department due to intermittent hematuria, left flank pain, and lower urinary tract symptoms that persisted for 2 weeks. His history was significant for undergoing left ureterolithotripsy followed by the implantation of an ipsilateral D-J stents 2 years ago in a local hospital. The patient did not follow-up regularly or actively seek medical attention for his urinary tract symptoms. Computed tomographic urography revealed a hyperdense tubular object protruding from the left distal ureter to the bladder. The patient underwent cystolithotripsy, left ureteric stent removal, and left ureteroscopy to clear away the bladder stone and its D-J stent core. CONCLUSIONS Formation of bladder stones secondary to prolonged indwelling D-J stent and its encrustation is not uncommon in developing countries where the level of public education is low. Prompt D-J stent removal can prevent complications associated with its retention and avoid unnecessary secondary procedures. Endoscopic urologic procedures are safe and feasible management options, and doctor-to-patient communication is vital for a better prognosis.

Risk factors for renal outcomes in children with antineutrophil cytoplasmic antibody-associated vasculitis: a nationwide retrospective study in China.

BACKGROUND: Pediatric antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is a life-threatening systemic vasculitis featured by liability to renal involvement. However, there are few studies on the risk factors and predictive models for renal outcomes of AAV in children. METHODS: Data from 179 AAV children in multiple centers between January 2012 and March 2020 were collected retrospectively. The risk factors and predictive model of end-stage renal disease (ESRD) in AAV were explored. RESULTS: Renal involvement was the most typical manifestation (95.5%), and the crescent was the predominant pathological lesion (84.9%). The estimated glomerular filtration rate (eGFR) was evaluated in 114 patients, of whom 59.6% developed ESRD, and the median time to ESRD was 3.20 months. The eGFR [P = 0.006, odds ratio (OR) = 0.955, 95% confidence interval (CI) = 0.924-0.987] and the percentages of global glomerulosclerosis (pGGS; P = 0.018, OR = 1.060, 95% CI = 1.010-1.112) were independent risk factors for ESRD of renal biopsy. Based on the pGGS and eGFR at renal biopsy, we developed three risk grades of ESRD and one predictive model. The Kaplan‒Meier curve indicated that renal outcomes were significantly different in different risk grades (P < 0.001). Compared with serum creatinine at baseline, the predictive model had higher accuracy (0.86 versus 0.58, P < 0.001) and a lower coefficient of variation (0.07 versus 0.92) in external validation. CONCLUSIONS: Renal involvement is the most common manifestation of pediatric AAV in China, of which more than half deteriorates into ESRD. The predictive model based on eGFR at renal biopsy and the pGGS may be stable and accurate in speculating the risk of ESRD in AAV children. Supplementary file 2 (MP4 18937 KB).

Canavalia gladiata Pod Extract Mitigates Ovalbumin-Induced Asthma Onset in Male BALB/c Mice via Suppression of MAPK.

Asthma is one of the most common inflammatory diseases of the lung worldwide. There has been considerable progress in recent studies to treat and prevent allergic asthma, however, various side effects are still observed in clinical practice. Six-week-old male BALB/c mice were orally administered with either sword bean pod extracts (SBP; 100 or 300 mg/kg) or dexamethasone (DEX; 5 mg/kg) once daily over 3 weeks, followed by ovalbumin sensitization (OVA/Alum.; intraperitoneal administration, 50 µg/2 mg/per mouse). Scoring of lung inflammation was performed to observe pathological changes in response to SBP treatment compared to OVA/Alum.-induced lung injury. Additionally, inflammatory cytokines were quantified in serum, bronchoalveolar lavage fluid (BALF), and lung tissue using ELISA and Western blot analyses. SBP treatment significantly reduced the infiltration of inflammatory cells, and release of histamine, immunoglobulin E, and leukotriene in serum and BALF. Moreover, the therapeutic effect of SBP was also assessed to analyze the inflammatory changes in the lung tissues. SBP markedly suppressed the activation of the MAPK signaling pathway and the expression of key inflammatory proteins (e.g., TNF-α) and Th2 type cytokines (IL-5 and IL-13). SBP was effective in ameliorating the allergic inflammation against OVA/Alum.-induced asthma by suppressing pulmonary inflammation.

Citrus junos Tanaka Peel Extract and Its Bioactive Naringin Reduce Fine Dust-Induced Respiratory Injury Markers in BALB/c Male Mice.

Particulate matter (PM) 10 refers to fine dust with a diameter of less than 10 µm and induces apoptosis and inflammatory responses through oxidative stress. Citrus junos Tanaka is a citrus fruit and contains bioactive flavonoids including naringin. In the present study, we aimed to identify the preventive effect of Citrus junos Tanaka peel extract (CPE) against PM10-induced lung injury. As a proof of concept, NCI-H460 cells were treated with CPE (800 µg/mL, 12 h) in conjunction with PM10 to examine intracellular antioxidative capacity in the pulmonary system. In an in vivo model, male BALB/c mice (n = 8/group) were randomly assigned into five groups: NEG (saline-treated), POS (PM10 only), NAR (PM10 + naringin, 100 mg/kg), CPL (PM10 + CPE low, 100 mg/kg), and CPH (PM10 + CPE high, 400 mg/kg). Intervention groups received dietary supplementations for 7 days followed by PM10 exposure (100 mg/kg, intranasal instillation). Compared to the NEG, the CPE decreased to 22% of the ROS generation and significantly increased cell viability in vitro. The histological assessments confirmed that pulmonary damages were alleviated in the PM10 + CPL group compared to the POS. Pro-inflammatory cytokines and NF-κB/apoptosis signaling-related markers were decreased in the PM10 + CPL group compared to the POS. These results indicated that CPE showed promising efficacy in preventing pulmonary injuries in vivo. Such protection can be explained by the anti-oxidative capacity of CPE, likely due to its bioactives, including naringin (7.74 mg/g CPE). Follow-up human intervention, as well as population-level studies, will further shed light on the preventive efficacy of CPE against pulmonary damage in humans.

Significance of Macrophage Subtypes in the Peripheral Blood of Children with Systemic Juvenile Idiopathic Arthritis.

INTRODUCTION: Symptomatic juvenile idiopathic arthritis (sJIA) is an autoinflammatory disease, and monocytes/macrophages play an important role. However, which macrophage subtype plays a major role in different stages of sJIA is still unclear. This study aimed to explore macrophage subtypes in different stages of sJIA. METHODS: Twenty-two children with sJIA who were followed up at Shanghai Children's Hospital from January 2018 to December 2020 were enrolled in this study. sJIA children were divided into an activity group (n = 12) and an inactivity group (n = 10). In the activity group, subjects with newly diagnosed sJIA and untreated were included; in the inactivity group, subjects with inactive sJIA meeting the 2011 ACR criteria for sJIA were recruited. Ten children with orthostatic proteinuria served as controls. Peripheral blood was collected. Flow cytometry was performed to detect macrophage subtypes: M1 (CD14+CD86+CD80+), M2a (CD14+CD206+CD301+), M2b (CD14+CD206+CD86+) and M2c (CD14+CD206+CD163+), and the contents of cytokines were also examined, including interleukins (IL) (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17), interferon-α, interferon-γ, and tumor necrosis-α. RESULTS: M1 marker CD80 and M2 marker CD163, CD301 were highly expressed in children with active sJIA. The majority of macrophages were M1 and M2a in the activity group (P < 0.05). In the inactivity group, M2 tended to polarize into M2b and M2c (P < 0.05). IL-6 significantly increased in the activity group (P < 0.05), while IL-10, IL-4 and IL-17 markedly increased in the inactivity group (P < 0.05). CONCLUSIONS: In the active sJIA, M1 activation promotes inflammation, while M2a rapidly responds to inhibit inflammation; in the inactive sJIA, M2b and M2c play a major role in inhibiting inflammation.

Generation of an induced pluripotent stem cell line (SHCDNRi001-A) from a patient with X-linked Alport syndrome carrying a heterozygous p.G409S (c. 1225 G > A) mutation in the COL4A5 gene.

X-linked Alport syndrome (XLAS) is a rare form of hereditary nephritis caused by mutations in the COL4A5 gene encoding the type IV collagen α5 chain. A skin biopsy was performed on one female patient with XLAS who carried a heterozygous p.G409S (c. 1225 G > A) mutation in the COL4A5 gene. A human-induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts using the integrating free Sendai virus technique. The generated iPSC line SHCDNRi001-A offers an efficient resource to research pathogenic mechanisms in XLAS, as well as a cell-based disease model for drug testing or other treatments.

Renal proteomic analysis of RGC-32 knockout mice reveals the potential mechanism of RGC-32 in regulating cell cycle.

This study aimed to investigate the exact function of RGC-32 in kidney diseases and explore the potential mechanism of RGC-32 in regulating cell cycle. RGC-32 knockout (RGC-32-/-) mice were generated from C57BL/6 embryonic stem cells. Differentially expressed proteins in the kidney were investigated with the isobaric tags for relative and absolute quantification (iTRAQ) technique. Gene ontology analyses (GO), Kyoto encyclopedia of genes and genomes (KEGG) pathway mapping analysis and functional network analysis were also performed. The expressions of Smc3, Smad 2-3, DNA-PK were further confirmed by qPCR. Results showed that 4690 proteins were quantified on the basis of 25165 unique peptides. Comparative proteomic analysis revealed 361 differentially expressed proteins in RGC-32-/- mice (knockout/wild ratio >+/- 1.2 and P<0.05). GO and KEGG pathway mapping analyses showed differentially expressed proteins were involved in spliceosome, fluid shear stress and atherosclerosis protein processing in endoplasmic reticulum, pathways in cancer, viral carcinogenesis, epithelial cell signaling in Helicobacter pylori infection, HTLV-I infection, PI3K-Akt signaling pathway, ubiquitin mediated proteolysis, Parkinson's disease, MAPK signaling pathway, carbon metabolism, Alzheimer's disease, NOD-like receptor signaling pathway, tight junction, Proteoglycans in cancer, phagosome, ribosome, mTOR signaling pathway, and AMPK signaling pathway. Differentially expressed proteins Smc3 (0.821), DNA-PK (0.761), Smad 2-3 (0.631) were involved in cell cycle regulation. mRNA expression of Smad2-3, DNA-PK, and Smc3 was consistent with that from iTRAQ. It is concluded that RGC-32 may affect the expression of many proteins (76 up-regulated and 285 down-regulated) in the kidney, and may regulate the expression of Smc3, DNA-PK and Smad 2-3 to affect the cell cycle.

Chylopericardium in a child with IgA nephropathy: a case report.

BACKGROUND: Chylopericardium effusion is characterized by the accumulation of milky effusion in the pericardium. It is often idiopathic but it can be secondary to trauma, chest radiation, tuberculosis and malignancy. If cardiac tamponade ensues, it becomes life-threatening. Herein we describe chylopericardium tamponade in a child with IgA nephropathy. To the best of our knowledge, this is the first reported case of chylopericardium tamponade in IgA nephropathy. CASE PRESENTATION: A 6 years old boy with IgA nephropathy presented with dyspnea, orthopnea, pretibial pitting edema, ascites and fever. Muffled heart sounds and hepatomegaly were also noted. Echocardiography and thoracic CT revealed that there was a large volume of hydropericardium. Moreover, the pericardial milky fluid by pericardiocentesis was analyzed and chylopericardium effusion was eventually confirmed. Pericardial drainage was continued and his diet was modified to low fat, rich MCT and high protein. Complete remission was achieved after 3 weeks of this combined treatment. CONCLUSION: Chylopericardial tamponade could be a rare and life-threatening complication of IgA nephropathy. Etiological analysis is critical for determining the therapeutic approach in patients with pericardial effusion.

The mechanism of epithelial-mesenchymal transition induced by TGF-ß1 in neuroblastoma cells.

Neuroblastoma is the second most common extracranial malignant solid tumor that occurs in childhood, and metastasis is one of the major causes of death in neuroblastoma patients. The epithelial-mesenchymal transition (EMT) is an important mechanism for both the initiation of tumor invasion and subsequent metastasis. Therefore, this study investigated the mechanism by which transforming growth factor (TGF)-ß1 induces EMT in human neuroblastoma cells. Using quantitative RT-qPCR and western blot analyses, we found that the mRNA and protein expression levels of E-cadherin were significantly decreased, whereas that of α-SMA was significantly increased after neuroblastoma cells were treated with different concentrations of TGF-ß1. A scratch test and Transwell migration assay revealed that cell migration significantly and directly correlated with the concentration of TGF-ß1 indicating that TGF-ß1 induced EMT in neuroblastoma cells and led to their migration. Inhibiting Smad2/3 expression did not affect the expression of the key molecules involved in EMT. Further investigation found that the expression of the glioblastoma transcription factor (Gli) significantly increased in TGF-ß1-stimulated neuroblastoma cells undergoing EMT, accordingly, interfering with Gli1/2 expression inhibited TGF-ß1-induced EMT in neuroblastoma cells. GANT61, which is a targeted inhibitor of Gli1 and Gli2, decreased cell viability and promoted cell apoptosis. Thus, TGF-ß1 induced EMT in neuroblastoma cells to increase their migration. Specifically, EMT induced by TGF-ß1 in neuroblastoma cells did not depend on the Smad signaling pathway, and the transcription factor Gli participated in TGF-ß1-induced EMT independent of Smad signaling.

A single-center analysis of Henoch-Schonlein purpura nephritis with nephrotic proteinuria in children.

BACKGROUND: In children with Henoch-Schonlein purpura nephritis (HSPN), the degree of proteinuria has been proven to be not only a sign of kidney damage, but also an accelerator of kidney disease progression. Nephrotic proteinuria at disease onset has been proposed as a predictor of a poor renal outcome. This study aims to assess the clinical and pathological features of HSPN with nephrotic proteinuria in a single center. METHODS: One hundred thirty-seven patients with HSPN who visited Shanghai Children's Hospital from January 2009 to December 2013 were retrospectively reviewed. The patients were divided into 2 groups based on the 24-h urinary protein levels: nephrotic proteinuria group (NP group: 24-h urinary protein ≥50 mg/kg) and non-nephrotic proteinuria group (NNP group: 24-h urinary protein <50 mg/kg). In addition, data regarding their sex, age, clinical features, renal pathology, and prognosis were collected. RESULTS: (1) There were 34 boys and 20 girls in the NP group with a mean age of 8.39 ± 2.85 years. The peak age of incidence was 6 to 11 years (72.22%). (2) There were 8 cases (14.81%) with joint symptoms and 9 cases (16.67%) with gastrointestinal symptoms in the NP group. According to the analysis of the laboratory test results, the serum albumin and IgG levels of the NP group were significantly lower than that of the NNP group (35.04 ± 8.45 in the NP group vs. 41.55 ± 4.46 in the NNP group, P < 0.0001; 7.68 ± 3.12 in the NP group vs. 9.53 ± 2.74 in the NNP group, P < 0.001, respectively); their blood urea nitrogen and cystatin C levels increased significantly (P < 0.05). (3) The majority of the pathological changes in the NP group were above the International Study of Kidney Disease in Children (ISKDC) grade III (62.97%). The NP group patients with tubulointerstitial injurie with grade 2 and above (50%) were prioritized. Immune complex deposition in the NP group was dominated by IgA. (4) The prognosis of the NP group was in complete remission (A), and their cases did not develop into end-stage renal disease; their prognosis was also associated with clinical classification (P < 0.01) but was not related to pathologic grading and tubulointerstitial injury (P > 0.05). CONCLUSION: The serum albumin and IgG levels of the NP group were significantly lower; however, their blood urea nitrogen and cystatin C levels were higher. The ISKDC grades were mainly above grade III. The prognosis of the NP group was associated with clinical classification and improved after a timely and early treatment.

Single dose of rituximab in children with steroid-dependent minimal change nephrotic syndrome.

Rituximab (RTX) can be used in children with nephrotic syndrome, particularly in those with steroid-dependent nephrotic syndrome (SDNS). However, at present there is no unified standard of how to use RTX, with regard to the amount of doses and frequency, in children with nephrotic syndrome. The study aimed to investigate the therapeutic efficacy of a single dose of RTX in children with steroid-dependent minimal change nephrotic syndrome (SD-MCNS). The patients with biopsy-proven minimal change disease (MCD) and clinical features of SDNS received a single dose of RTX (375 mg/m2). The toxicity and side effects of RTX were also observed. The study included 19 patients (10 males and 9 females). Follow-up of the patients was 1-50 months (28.1±16.6 months). B-cell depletion was achieved with RTX infusion (CD20<0.5%) and lasted 1-6 months (mean, 2.92±1.57 months). During follow-up, 10 patients remained in complete remission and did not relapse without administration of oral steroids or immunosuppressants for 4-50 months (mean, 30.1±12.6 months), despite recovery of the B-cell count. Nine patients relapsed in the process of reducing steroids, thus, treatment was maintained at a lower dosage (T=0, P<0.05) than prior to use of RTX. The number of relapses also decreased significantly (T=95, P<0.05). Five of the patients relapsed after stopping steroid for several months. At the end of follow-up, the efficacy of a single induction of RTX was 47.4% (9/19). There were no significant side effects associated with administration of RTX. In conclusion, RTX is a safe and effective alternative for children with SD-MCNS. RTX is an effective treatment for the rapid induction of remission and reduces relapse and steroid dependency. A single dose of RTX for children with SD-MCNS is recommended for rapid induction of remission, reduction of long-term steroid dosage, and decrease in the number of relapses, as it has few side effects.

Response gene to complement 32 regulates the G2/M phase checkpoint during renal tubular epithelial cell repair.

BACKGROUND: The aim of this study was to evaluate the influence of RGC-32 (response gene to complement 32) on cell cycle progression in renal tubular epithelial cell injury. METHODS: NRK-52E cells with overexpressed or silenced RGC-32 were constructed via transient transfection with RGC-32 expression plasmid and RGC-32 siRNA plasmid, and the cell cycle distribution was determined. The expression levels of fibrosis factors, including smooth muscle action (α-SMA), fibronectin (FN) and E-cadherin, were assessed in cells with silenced RGC-32. RESULTS: The cells were injured via TNF-α treatment, and the injury was detectable by the enhanced expression of neutrophil gelatinase-associated lipocalin (NGAL). RGC-32 expression also increased significantly. The number of cells at G2/M phase increased dramatically in RGC-32 silenced cells, indicating that RGC-32 silencing induced G2/M arrest. In addition, after treatment with TNF-α, the NRK-52E cells with silenced RGC-32 showed significantly increased expression of α-SMA and FN, but decreased expression of E-cadherin. CONCLUSIONS: The results of this study suggest that RGC-32 probably has an important impact on the repair process of renal tubular epithelial cells in vitro by regulating the G2/M phase checkpoint, cell fibrosis and cell adhesion. However, the exact mechanism needs to be further elucidated.

A single center clinical analysis of children with neuroblastoma.

In the present study, the cases of 59 children diagnosed with neuroblastoma (NB) were retrospectively analyzed to assess the association between the short-term efficacy of treatment and prognostic factors. In total, 59 patients with NB that were diagnosed between July 1, 2008 and June 30, 2013 at Shanghai Children's Hospital were enrolled in the present study. The follow-up was performed until December 31, 2013, and the data revealed that 43 patients (72.9%) achieved complete remission (CR) or partial remission (PR). The 3-year overall survival (OS) rate of patients with stage I, II, III, IV and IVs disease was 100, 100, 65.6, 34.8 and 85.7%, respectively (P=0.02). The 3-year OS and event-free survival rates were evidently increased in patients with favorable histology compared with the rates in the patients with unfavorable histology (P=0.046 and 0.030, respectively). Univariate statistical analysis revealed that the factors significantly associated with prognosis were patient age, tumor stage and risk group (P=0.004, 0.02 and 0.001, respectively). The present study identified that tumor stage, risk group and patient age are important prognostic factors for NB. An age of 18 months was also hypothesized to be the cut-off for the prognosis of patients.

Expression of response gene to complement-32 in renal tissue of children with immunoglobulin A nephropathy.

OBJECTIVE: This study aimed to investigate the expression and significance of response gene to complement-32 (RGC-32) in renal tissue of children with immunoglobulin A nephropathy (IgAN). MATERIAL AND METHODS: Forty-five patients diagnosed as having IgAN by renal biopsy were enrolled. The expression of RGC-32, α-smooth muscle actin (α-SMA) and transforming growth factor-ß(1) (TGF-ß(1)) was observed by immunohistostaining. The relationshis between the expression of RGC-32, α-SMA, TGF-ß1, degree of renal pathological lesions in IgAN and clinical index were assessed by Spearman correlation. RESULTS: Immunohistostaining analysis showed that RGC-32 protein was present in epithelial cells of renal tubules in normal and IgAN renal tissues. With more severe renal pathological lesions, the expression of RGC-32 in IgAN was increased. The expression of RGC-32 was positively correlated with that of α-SMA, TGF-ß(1) and the degree of renal pathological lesions in children with IgAN (p < 0.05), but had no relationship with serum creatinine, urinary N-acetyl-ß-d-glucosaminidase/creatinine, urinary microalbuminuria/creatinine, urinary microimmunoglobulin/creatinine or urinary α(1)-microglobulin/creatinine ratio (p > 0.05). CONCLUSION: Expression of RGC-32 can reflect the degree of renal pathological lesions in IgAN. RGC-32 may participate in the renal tubulointerstitial lesions in children with IgAN, especially in epithelial -mesenchymal transition induced by TGF-ß(1).

Smad2 and PEA3 cooperatively regulate transcription of response gene to complement 32 in TGF-ß-induced smooth muscle cell differentiation of neural crest cells.

Response gene to complement 32 (RGC-32) is activated by transforming growth factor- ß (TGF-ß) and plays an important role in smooth muscle cell (SMC) differentiation from neural crest Monc-1 cells. The molecular mechanism governing TGF-ß activation of RGC-32, however, remains to be determined. The present studies indicate that TGF-ß regulates RGC-32 gene transcription. Sequence analysis revealed a Smad binding element (SBE) located in the region from -1344 to -1337 bp upstream of the transcription start site of RGC-32 gene. A polyomavirus enhancer activator (PEA3) binding site is adjacent to the SBE. Mutation at either SBE or PEA3 site significantly inhibited RGC-32 promoter activity. Mutations at both sites completely abolished TGF-ß-induced promoter activity. Biochemically, TGF-ß stimulated recruitment of Smad2, Smad4, and PEA3 to the RGC-32 promoter, as revealed by gel shift and chromatin immunoprecipitation analyses. Functionally, Smad2, but not Smad3, activated RGC-32 promoter. PEA3 appeared to enhance Smad2 activity. In agreement with their function, Smad2, but not Smad3, physically interacted with PEA3. In TGF-ß-induced SMC differentiation of Monc-1 cells, knockdown of Smad2 by short hairpin RNA resulted in downregulation of RGC-32 and SMC marker genes. The downregulation of SMC markers, however, was rescued by exogenously introduced RGC-32. These results demonstrate that Smad2 regulation of RGC-32 transcription is essential for SMC differentiation from neural crest cells.

RGC-32 mediates transforming growth factor-beta-induced epithelial-mesenchymal transition in human renal proximal tubular cells.

Epithelial-mesenchymal transition (EMT) occurs in several disease states, including renal fibrosis and carcinogenesis. Myofibroblasts produced from EMT of renal tubular cells are responsible for the deposition of extracellular matrix components in a large portion of renal interstitial fibrosis. Transforming growth factor-beta (TGF-beta) plays an essential role in the EMT of renal tubular cells, but the molecular mechanism governing this process remains largely unknown. In this study, we found that RGC-32 (response gene to complement 32) is critical for TGF-beta-induced EMT of human renal proximal tubular cells (HPTCs). RGC-32 is not normally expressed in the HPTCs. However, TGF-beta stimulation markedly activates RGC-32 while inducing an EMT, as shown by the induction of smooth muscle alpha-actin (alpha-SMA) and extracellular matrix proteins collagen I and fibronectin, as well as the reduction of epithelial marker E-cadherin. TGF-beta function is mediated by several signaling pathways, but RGC-32 expression in HPTCs appears to be mainly regulated by Smad. Functionally, RGC-32 appears to mediate TGF-beta-induced EMT of HPTCs. Blockage of RGC-32 using short hairpin interfering RNA significantly inhibits TGF-beta induction of myofibroblast marker gene alpha-SMA while repressing the expression of E-cadherin. In contrast, overexpression of RGC-32 induces alpha-SMA expression while restoring E-cadherin. RGC-32 also inhibits the expression of another adherens junction protein, N-cadherin, suggesting that RGC-32 alone induces the phenotypic conversion of renal epithelial cells to myofibroblasts. Additional studies show that RGC-32 stimulates the production of extracellular matrix components fibronectin and collagen I. Mechanistically, RGC-32 induces EMT via the activation of other transcription factors such as Snail and Slug. RGC-32 knockdown inhibits the expression of Snail and Slug during TGF-beta-induced EMT. Taken together, our data demonstrate for the first time that RGC-32 plays a critical role in TGF-beta-induced EMT of renal tubular cells.

[Prohibitin suppresses renal interstitial fibroblasts proliferation and phenotypic change induced by transforming growth factor-beta1].

OBJECTIVE: To illuminate the possible role of Prohibitin (PHB) in tubulointerstitial fibrosis. METHODS: (1) Forty-eight renal biopsy specimens were obtained from the patients with various primary glomerulonephritis, 26 male and 22 female, aged 7.5 +/- 5.5 (2.5 - 13 years), and nine kidney tissue specimens were obtained form the tissues far away from the tumor tissues and confirmed by pathological examination as normal tissues in the kidney dissected during operation as normal control. Immunohistochemistry was used to detect the protein expression of PHB and alpha-smooth muscle actin (alpha-SMA). The correlation between PHB and degree of tubulointerstitial lesion was compared. (2) Rat kidney fibroblastoma cells of the line NRK-49F were cultured, and laser scanning confocal microscopy was used to observe the subcellular location of PHB protein. The changes of PHB protein and mRNA expression in the NRK-49F cells upon TGF-beta1 stimulation were detected by Western blotting and RT-PCR analysis. (3) PHB expression plasmid was constructed and transfected into the NRK-49F cells. Then, cell cycle analysis was performed by flow cytometry, and Western blotting and RT-PCR were performed to detect the PHB and alpha-SMA protein and mRNA expression in the NRK-49F cells treated with or without TGF-beta1. RESULTS: (1) PHB protein expression was found in the normal renal tissues by immunohistochemistry, with a positive distribution in the interstitial cells and tubular epithelial cells. PHB was strongly down-regulated in the damaged interstitial and tubular epithelial cells, the higher the grade of damage, the lower the expression of PHB (all P < 0.01), and the PHB expression amount was negatively correlated with the degree of tubulointerstitial lesions (r = -0.802, P < 0.01). (2) Confocal microscopy showed that PHB was mainly located in the cytoplasm and weakly expressed in the nucleus of the NRK-49F cells. Treated with TGF-beta1, the PHB protein expression and mRNA expression in the NRK-49F cells were decreased both time-dependently and dose-dependently (all P < 0.01). (3) A recombinant pcDNA3.1 (-)/PHB plasmid was successfully constructed. PHB protein expression in the transfected NRK-49F cells was 2.54 times higher compared with the non-transfected cells. (4) The proportions of the cells in the S and G(2)/M phases were higher in the NRK-49F cells stimulated by TGF-beta1, however, more NRK-49F cells remained in the G(0)/G(1) phase after transfection of PHB (P < 0.01). (5) Both alpha-SMA protein and mRNA were not expressed in the control cells while de novo expression of alpha-SMA in the NRK-49F cells was increased after the treatment of TGF-beta1. Over-expression of PHB did not affect he basic alpha-SMA expression but dramatically repressed TGF-beta1-initiated alpha-SMA expression in the NRK-49F cells (P < 0.01). CONCLUSION: PHB protein is expressed in the normal renal tissues and adversely correlated with the degree of tubulointerstitial lesions. Extraneous PHB suppresses renal interstitial fibroblast proliferation and cell phenotypic change induced by TGF-beta1.

Prohibitin suppresses renal interstitial fibroblasts proliferation and phenotypic change induced by transforming growth factor-beta1.

Prohibitin (PHB), a potential tumor suppressor, has been shown to inhibit cell proliferation by repressing E2F-mediated transcription. But little is known about the role of PHB involved in tubulointerstitial fibrosis (TIF). Here, for the first time, we found PHB protein was positively expressed at normal renal tissues, strongly down-regulated in renal biopsy specimens, and negatively correlated with the expression of alpha-smooth-muscle actin (alpha-SMA) and with the degrees of tubulointerstitial lesions. Transforming growth factor-beta1 (TGF-beta1) is the most important profibrotic cytokine in the process of TIF and capable of inducing cell phenotypic change of interstitial fibroblasts characterized by the de novo expression of alpha-SMA. Confocal microscopy showed majority of PHB is located at cytoplasm as well as at nucleus in rat kidney fibroblasts cell (NRK-49F). As we found that PHB protein and mRNA expression were down-regulated in NRK-49F cells following TGF-beta1 stimulation. We used transient transfection to over-express PHB protein and found that cells with increased PHB levels had a significant reduction in the percentage entering cell cycle and abolished de novo expression of alpha-SMA following TGF-beta1 stimulation. Therefore, over-expression of PHB suppresses renal interstitial fibroblasts proliferation and cell phenotypic change induced by TGF-beta1, which indicates PHB as a potential therapeutic target to halt the progression of TIF.