MicroRNA-26a-5p Restoration Ameliorates Unilateral Ureteral Obstruction-Induced Renal Fibrosis in Mice Through Modulating TGF-ß Signaling.

Renal fibrosis is a hallmark of chronic and progressive renal diseases characterized by excessive fibroblast proliferation, extracellular matrix accumulation, and a loss of renal function, eventually leading to end-stage renal diseases. MicroRNA-26a-5p (miR-26a-5p) downregulation has been previously noted in the sera of unilateral ureteral occlusion (UUO)-injured mice, and exosome-mediated miR-26a-5p reportedly attenuated experimental pulmonary and cardiac fibrosis. This study evaluated the expression patterns of miR-26a in a human tissue microarray with kidney fibrosis and in tissues from a mouse model of UUO-induced renal fibrosis. Histologic analyses showed that miR-26a-5p was downregulated in human and mouse tissues with renal interstitial nephritis and fibrosis. Moreover, miR-26a-5p restoration by intravenous injection of a mimic agent prominently suppressed the expression of transforming growth factor ß1 (TGF-ß1) and its cognate receptors, the inflammatory transcription factor NF-κB, epithelial-mesenchymal transition, and inflammatory markers in UUO-injured kidney tissues. In vitro, miR-26a-5p mimic delivery significantly inhibited TGF-ß1-induced activation of cultured normal rat kidney NRK-49F cells, in terms of downregulation of TGF-ß1 receptors, restoration of the epithelial marker E-cadherin, and suppression of mesenchymal markers, including vimentin, fibronectin, and α-smooth muscle actin, as well as TGF-ß1/SMAD3 signaling activity. Our findings identified miR-26a-5p downregulation in kidney tissues with human interstitial nephritis and UUO-induced mouse kidney fibrosis. MiR-26a-5p restoration may exhibit an antifibrotic effect through the blockade of both TGF-ß and NF-κB signaling axes and is considered a novel therapeutic target for treating obstruction-induced renal fibrosis.

Genetic Interference of FGFR3 Impedes Invasion of Upper Tract Urothelial Carcinoma Cells by Alleviating RAS/MAPK Signal Activity.

Upper tract urothelial cancer (UTUC) is a less common disease in Western countries but has a high level of prevalence in Asian populations. Compared to bladder cancer, unique etiologic and genomic factors are involved in UTUC. Fibroblast growth factor receptor 3 (FGFR3) up-regulation has been proposed as a promising target for bladder cancer therapy. In this study, we aimed to profile the expression of FGFR3 in Asian and Caucasian UTUC tissues and to evaluate the in vitro therapeutic efficacy of small interference RNA (siRNA)-mediated FGFR3 silencing in UTUC treatment. The FGFR3 expression levels in renal pelvis tissues and microarray sections from Asian and Caucasian patients with UTUC, respectively, were measured via immunohistochemistry. The BFTC-909 and UM-UC-14 UTUC cell lines were used to examine the effects of FGFR3 silencing on proliferation, migration, epithelial-mesenchymal transition (EMT) marker expression, and signaling machinery. FGFR3 expression increased as the TNM stage increased in both Asian and Caucasian UTUC tumors, and no statistical difference was identified between the two groups. In vitro studies demonstrated that FGFR3 siRNA delivery significantly inhibited proliferation and migration and suppressed the expression of EMT markers and transcription factors in UTUC cells. Mechanistically, FGFR3 silencing alleviated the constitutive expression of RAS and the phosphorylation of MAPK signaling mediators, including ERK1/2 and JNK1/2. FGFR3 silencing elicited an apoptosis-inducing effect similar to that of FGFR inhibition. Conclusion: siRNA-targeted FGFR3 expression may impede the expansion and invasion of UTUC cells by alleviating the RAS/MAPK signaling pathway. The genetic interference of FGFR3 expression via siRNA in UTUC cells may constitute a useful therapeutic strategy.

Urinary Uromodulin/Creatinine Ratio as a Potential Clinical Biomarker for Chronic Kidney Disease Patients with Gout: A Pilot Study.

OBJECTIVE: Recent studies have reported that reduced excretion of urinary uromodulin is associated with renal tubular function and risks of progressive kidney disease. Gouty nephropathy is usually seen in patients with gout. Patients with chronic gouty nephropathy are characterized by the deposition of monosodium urate crystals primarily involving the collecting ducts in the medulla. We postulated that this correlation may be specific to gout and may serve as a useful biomarker for chronic kidney disease (CKD). MATERIALS AND METHODS: A total of 114 Taiwanese patients diagnosed with gout (n = 72), CKD (n = 26), or healthy volunteers (n = 16) were prospectively enrolled for this study from the Rheumatology and Nephrology Outpatient Clinics of our institution. We obtained urine and blood samples on patient visits to the outpatient clinics. Demographic data were obtained from medical records. RESULTS: In patients with gout, the spot urinary uromodulin/creatinine ratio (uUMCR; mg/g) in patients with CKD was significantly lower than that in those without CKD (CKD group: 2.2; non-CKD group: 5.6, p = 0.005). Multivariate analysis revealed that patients with CKD and gout had a lower uUMCR than those with gout alone (p = 0.028). A significant association was not observed in our non-gout cohort. CONCLUSION: The association of decreased uUMCR with CKD status was identified only in patients with gout in the present study. We believe that uUMCR might serve as an indicator of differential CKD in patients with gout.

BMP-2 restoration aids in recovery from liver fibrosis by attenuating TGF-ß1 signaling.

Transforming growth factor-ß (TGF-ß) plays a central role in hepatic fibrogenesis. This study investigated the function and mechanism of bone morphogenetic protein-2 (BMP-2) in regulation of hepatic fibrogenesis. BMP-2 expression in fibrotic liver was measured in human tissue microarray and mouse models of liver fibrosis induced by bile duct ligation surgery or carbon tetrachloride administration. Adenovirus-mediated BMP-2 gene delivery was used to test the prophylactic effect on liver fibrosis. Primary hepatic stellate cells (HSC), HSC-T6 and clone-9 cell lines were used to study the interplay between BMP-2 and TGF-ß1. Hepatic BMP-2 was localized in parenchymal hepatocytes and activated HSCs and significantly decreased in human and mouse fibrotic livers, showing an opposite pattern of hepatic TGF-ß1 contents. BMP-2 gene delivery alleviated the elevations of serum hepatic enzymes, cholangiocyte marker CK19, HSC activation markers, and liver fibrosis in both models. Mechanistically, exogenous TGF-ß1 dose dependently reduced BMP-2 expression, whereas BMP-2 significantly suppressed expression of TGF-ß and its cognate type I and II receptor peptides, as well as the induced Smad3 phosphorylation levels in primary mouse HSCs. Aside from its suppressive effects on cell proliferation and migration, BMP-2 treatment prominently attenuated the TGF-ß1-stimulated α-SMA and fibronectin expression, and reversed the TGF-ß1-modulated epithelial-to-mesenchymal transition marker expression in mouse HSCs. The mutual regulation between BMP-2 and TGF-ß1 signaling axes may constitute the anti-fibrogenic mechanism of BMP-2 in the pathogenesis of liver fibrosis. BMP-2 may potentially serve as a novel therapeutic target for treatment of liver fibrosis.

MiR-30a-5p Inhibits Epithelial-to-Mesenchymal Transition and Upregulates Expression of Tight Junction Protein Claudin-5 in Human Upper Tract Urothelial Carcinoma Cells.

The involvement of microRNAs (miRNAs) in cancer development and their potential as prognostic biomarkers are becoming increasingly known. However, the signature of miRNAs and their regulatory roles in tumorigenesis of upper tract urothelial carcinoma (UTUC) remain to be elucidated. This study aimed to profile the miRNA expression pattern in UTUC tumor tissues and identify candidate miRNAs with prognostic and/or therapeutic functions. METHODS AND RESULTS: We collected 22 UTUC tissue and adjacent normal tissues samples from patients who underwent nephroureterectomy. The miRNAs signatures of three selected UTUC samples using next-generation sequencing showed that miR-30a-5p was significantly downregulated in UTUC tumors compared to adjacent normal tissues. The differentially-expressed miRNAs were specifically validated by quantitative real-time polymerase chain reaction. In addition, the miRNA expression signatures were analyzed with the transcriptome profile characterized by microarray. Further in vitro studies indicated that overexpression of miR-30a-5p significantly suppressed proliferation, migration, and epithelial-to-mesenchymal transition (EMT) in cultured BFTC-909 UTUC cells. As a potential target gene of miR-30a-5p in the tight junction pathway suggested by the pathway enrichment analysis, the reduced expression of tight junction protein claudin-5 in UTUC cells was demonstrated to be upregulated by miR-30a-5p genetic delivery. CONCLUSIONS: Taken together, our findings demonstrated that miR-30a-5p inhibits proliferation, metastasis, and EMT, and upregulates the expression of tight junction claudin-5 in UTUC cells. Thus, miR-30a-5p may provide a promising therapeutic strategy for UTUC treatment.

MiR-26a-5p as a useful therapeutic target for upper tract urothelial carcinoma by regulating WNT5A/ß-catenin signaling.

The role of miRNAs in cancer and their possible function as therapeutic agents are interesting and needed further investigation. The miR-26a-5p had been demonstrated as a tumor suppressor in various cancers. However, the importance of miR-26a-5p regulation in upper tract urothelial carcinoma (UTUC) remains unclear. Here, we aimed to explore the miR-26a-5p expression in UTUC tissues and to identify its regulatory targets and signal network involved in UTUC tumorigenesis. The miR-26a-5p expression was validated by quantitative real-time polymerase chain reaction (qPCR) using renal pelvis tissue samples from 22 patients who were diagnosed with UTUC and 64 cases of renal pelvis tissue microarray using in situ hybridization staining. BFTC-909 UTUC cells were used to examine the effects of miR-26a-5p genetic delivery on proliferation, migration and expression of epithelial-to-mesenchymal transition (EMT) markers. MiR-26a-5p was significantly down-regulated in UTUC tumors compared to adjacent normal tissue and was decreased with histological grades. Moreover, restoration of miR-26a-5p showed inhibition effects on proliferation and migration of BFTC-909 cells. In addition, miR-26a-5p delivery regulated the EMT marker expression and inhibited WNT5A/ß-catenin signaling and expression of downstream molecules including NF-κB and MMP-9 in BFTC-909 cells. This study demonstrated that miR-26a-5p restoration may reverse EMT process and regulate WNT5A/ß-catenin signaling in UTUC cells. Further studies warranted to explore the potential roles in biomarkers for diagnostics and prognosis, as well as novel therapeutics targets for UTUC treatment.

Upregulation of hepatoma-derived growth factor is involved in murine hepatic fibrogenesis.

BACKGROUND & AIMS: Hepatoma-derived growth factor (HDGF) expression is correlated with progression of hepatocellular carcinoma. Since liver fibrosis frequently occurs before hepatoma development, this study investigated the expression profile of HDGF and its relationship with transforming growth factor-beta (TGF-beta) signaling in experimental models of hepatofibrogenesis. METHODS: Liver fibrosis was induced in mice receiving bile duct ligation (BDL) or carbon tetrachloride (CCl(4)) administration. The expression levels of HDGF and other fibrosis-related markers were measured using quantitative RT-PCR, Western blotting, and enzyme-linked immunosorbent assays. Hepatic HDGF overexpression was achieved by adenovirus gene delivery. Rat hepatocytes were used to study the interplay between HDGF and TGF-beta1. RESULTS: In both liver fibrosis models, HDGF de novo synthesis significantly increased during the progression of fibrosis. The HDGF upregulation was observed mainly in hepatocytes and correlated with the expression of TGF-beta1 and collagen COL1A1 and COL1A2 proteins. Hepatic HDGF overexpression itself deteriorated hepatocellular structure and integrity, and aggravated the extents of BDL- and CCl(4)-induced liver fibrosis with concomitant upregulation of TGF-beta1 and COL1A1. Exogenous TGF-beta1 stimulated HDGF expression only in cultured primary hepatocytes grown on collagen matrix, whereas exogenous HDGF also increased TGF-beta1 production in hepatocytes in a collagen-dependent manner. Moreover, HDGF enhanced Smad2 phosphorylation dose-dependently and the TGF-beta1-driven luciferase activities. CONCLUSION: HDGF plays a pro-fibrogenic role during liver fibrosis in mice through activation of TGF-beta pathway. The mutual regulation between TGF-beta1 and HDGF may facilitate a vicious cycle to promote the progression of hepatic fibrogenesis.

Vulnerability of the spinal cord to injury from extracorporeal shock waves in rabbits.

We studied the vulnerability of the spinal cord to extracorporeal shock wave treatment (ESWT). In this experiment, 12 rabbits were divided into three groups (4 in each group). All animals underwent a preceding lumbar laminectomy at L4 1 week before ESWT. In group 1, 2000 impulses of high dose (0.62 mJ/mm2 energy flux density) shockwave energy were applied to the spinal cord at the laminectomy site. In group 2, 2000 impulses of low dose (0.18 mJ/mm2 energy flux density) shockwave energy were applied to the same site as group 1. Group 3 did not receive ESWT and served as a control. None of the rabbits in the study groups (groups 1 and 2) showed weakness or paralysis of the hind limbs throughout the entire post-ESWT period. The spinal cord at the L4 level of all animals was harvested on day 13 after laminectomy. On gross morphology, the cord from the study groups and the control group showed normal surface appearance. On microscopic examination, the cord from the control group was normal, whereas the cords from the study groups showed varying degrees of myelin damage and neuronal loss. These microscopic findings were dose-dependent. For the low-energy group (group 2), neuronal loss was insignificant compared to that in the control group. ESWT produced varying degrees of microscopic changes of the treated cords, but no neurological symptoms. The neuronal injury was dose-dependent and mild in the low-energy group.

Expression of Estrogen Receptor Beta Predicts Oncologic Outcome of pT3 Upper Urinary Tract Urothelial Carcinoma Better Than Aggressive Pathological Features.

Upper urinary tract urothelial carcinoma (UT-UC) is rare and treatment options or prognostic markers are limited. There is increasing evidence indicating that urothelial carcinoma may be an endocrine-related cancer. The aim of this study was to analyze the prognostic effect of estrogen receptor beta (ERß) on the outcome of UT-UC. From 2005 to 2012, this study included 105 patients with pT3 UT-UC. Perioperative factors, pathological features, and ERß immunostaining were reviewed and prognostic effects were examined by multivariate analysis. This study divided patients into either the ERß-high (n = 52) or ERß-low (n = 53) group and analyzed their oncologic outcomes. All pathological features except infiltrating tumor architecture (significantly higher incidence in ERß-low group, p = 0.004) are symmetric in both groups. Low ERß expression was significantly correlated with local recurrence and distant metastasis in univariate analysis (p = 0.035 and 0.004, respectively) and multivariate analysis (p = 0.05 and 0.008, respectively). Cell line study also proved that knock down of ERß cause less UTUC proliferation and migration. In addition, ERß agonist also enhanced the cytotoxic and migration inhibition effect of cisplatin and ERß antagonist cause the UTUC cell more resistant to cisplatin. This result may help identify patients in need of adjuvant therapy or develop potential targeted therapy.

Development of hyperthyroidism following primary hypothyroidism: a case report.

Development of hyperthyroidism following primary hypothyroidism is uncommon, and only a few documented cases have been reported. Alterations in thyroid-stimulating hormone receptor antibodies in serum are currently considered to play the main role in the pathophysiology, but the exact mechanism is still unknown. Here, we report the case of a 60-year-old man with disturbed consciousness due to hyponatremia. Thyroid function tests showed primary hypothyroidism with a high anti-microsomal antibody titer (1:6,400). The patient experienced weight loss and exophthalmos 6 years later. Serum thyroid hormone levels were increased and thyroxine treatment was discontinued, but the patient remained thyrotoxic 2 months later. 131I thyroid uptake was 40.9% at 24 hours, and bilateral thyroid lobes were not enlarged with diffuse radioactivity. Six months later, the patient was still thyrotoxic and therapy with methimazole 10 mg/day was started. He is now taking methimazole and is euthyroid.

OnobotulinumtoxinA Has No Effects on Growth of LNCaP and PC3 Human Prostate Cancer Cells.

OBJECTIVES: Intraprostatic injection of botulinum toxin (BTX) has been reported to have therapeutic effects on lower urinary tract symptoms related to benign prostate hyperplasia (BPH). Patients with BPH are at risk of having prostate cancer. The present study was conducted to assess the effect of onobotulinumtoxinA on prostate cancer in vitro and in vivo. METHODS: Human prostate cancer cell lines, LNCaP and PC3 were exposed to different doses of onobotulinumtoxinA (0-10 U; Allergan, Irvine, CA, USA). Cell viability, DNA fragmentation and apoptosis assay were subsequently measured. For the in vivo study, LNCaP and PC3 xenografts were introduced into nude mice and treated with intra-tumoral onobotulinumtoxinA or saline injection. Tumor volume, histopathology and apoptosis were assessed. Presence of SNAP-25 protein, the molecular target of onobotulinumtoxinA, was studied in both cell lines by Western blot analysis. RESULTS: OnobotulinumtoxinA did not significantly affect cell proliferation or apoptosis in LNCaP and PC3 cells. There was no significant difference in tumor size and histopathological findings between the experimental and control groups. There was no detectable SNAP-25 protein in both cell lines. CONCLUSION: OnobotulinumtoxinA does not affect the growth of LNCaP or PC3 cells in vitro and in vivo or produce significant anti-tumor effects. Intraprostatic BTX injection for BPH might not affect the growth of prostate cancer.

Sequence-based polymorphisms in the mitochondrial D-loop and potential SNP predictors for chronic dialysis.

BACKGROUND: The mitochondrial (mt) displacement loop (D-loop) is known to accumulate structural alterations and mutations. The aim of this study was to investigate the prevalence of single nucleotide polymorphisms (SNPs) within the D-loop among chronic dialysis patients and healthy controls. METHODOLOGY AND PRINCIPAL FINDINGS: We enrolled 193 chronic dialysis patients and 704 healthy controls. SNPs were identified by large scale D-loop sequencing and bioinformatic analysis. Chronic dialysis patients had lower body mass index, blood thiols, and cholesterol levels than controls. A total of 77 SNPs matched with the positions in reference of the Revised Cambridge Reference Sequence (CRS) were found in the study population. Chronic dialysis patients had a significantly higher incidence of 9 SNPs compared to controls. These include SNP5 (16108Y), SNP17 (16172Y), SNP21 (16223Y), SNP34 (16274R), SNP35 (16278Y), SNP55 (16463R), SNP56 (16519Y), SNP64 (185R), and SNP65 (189R) in D-loop of CRS. Among these SNPs with genotypes, SNP55-G, SNP56-C, and SNP64-A were 4.78, 1.47, and 5.15 times more frequent in dialysis patients compared to controls (P<0.05), respectively. When adjusting the covariates of demographics and comorbidities, SNP64-A was 5.13 times more frequent in dialysis patients compared to controls (P<0.01). Furthermore, SNP64-A was found to be 35.80, 3.48, 4.69, 5,55, and 4.67 times higher in female patients and in patients without diabetes, coronary artery disease, smoking, and hypertension in an independent significance manner (P<0.05), respectively. In patients older than 50 years or with hypertension, SNP34-A and SNP17-C were found to be 7.97 and 3.71 times more frequent (P<0.05) compared to patients younger than 50 years or those without hypertension, respectively. CONCLUSIONS AND SIGNIFICANCE: The results of large-scale sequencing suggest that specific SNPs in the mtDNA D-loop are significantly associated with chronic dialysis. These SNPs can be considered as potential predictors for chronic dialysis.

Bone morphogenetic protein gene therapy using a fibrin scaffold for a rabbit spinal-fusion experiment.

OBJECTIVE: To observe the feasibility of using fibrin gel as a scaffold during rabbit spinal fusion with rat kidney cells carrying bone morphogenetic protein-2 (BMP-2) expression vector. METHODS: Normal Rattusnorvegicus (rat) kidney (NRK) cells were transfected with pCMV-BMP2 vectors by using a nonviral reagent (FuGENE6), following which the transfected cells (1.5 x 10(7)) were encapsulated and evenly suspended in a fibrin scaffold, and implanted to either side of the L5-L6 intertransverse space for six test rabbits. For the control group (six rabbits), the transfected cells (1.5 x 10(7)) were added on and absorbed in a surgical gelatin sponge, and implanted in an analogous location. Radiographs of the spine of all animals were taken at six, ten, and 12 weeks subsequent to fusion surgery. Gross and histological examination of the fusion masses for all the animals were performed subsequent to animals having been sacrificed at 12 weeks. RESULTS: Expression of BMP-2 was verified in the pCMV-BMP2 transfected NRK cells which were used for the subsequent rabbit experiment. For all 12 rabbits, no evidence of implanted-tissue rejection was seen during the postoperative course. Neither residual scaffold nor inflammatory granulation tissue was seen in the harvested spinal segments. For the six study-group rabbits, radiographic examinations revealed that four individuals (67%) featured prominent new-bone formation (good fusion), and two (33%) revealed moderate new-bone formation (fair fusion) at the implanted sites, whereas all six control-group rabbits revealed no evidence of new-bone formation (nonfusion) at the implanted sites. For the histological examinations, all animals in the study group revealed the presence of osseous tissue at the sites corresponding to the sites of radiographically demonstrated new-bone formation, whereas for the control group, no osseous tissue was seen at the implanted sites for any animal CONCLUSION: Fibrin gel, being a biocompatible and biodegradable matrix, can encapsulate pCMV-BMP2 transfected NRK cells and adhere to the intertransverse lumbar-spine spaces of test rabbits. With the above characteristics, it plays an important role in the successful delivery of pCMV-BMP2-transfected cells for this rabbit spinal-fusion experiment. Being a natural matrix and able to be obtained readily from patients' own blood, fibrin gel seems to be a promising scaffold for future gene therapy in human trials.