LTBP4 Protects Against Renal Fibrosis via Mitochondrial and Vascular Impacts.

BACKGROUND: As a part of natural disease progression, acute kidney injury (AKI) can develop into chronic kidney disease via renal fibrosis and inflammation. LTBP4 (latent transforming growth factor beta binding protein 4) regulates transforming growth factor beta, which plays a role in renal fibrosis pathogenesis. We previously investigated the role of LTBP4 in chronic kidney disease. Here, we examined the role of LTBP4 in AKI. METHODS: LTBP4 expression was evaluated in human renal tissues, obtained from healthy individuals and patients with AKI, using immunohistochemistry. LTBP4 was knocked down in both C57BL/6 mice and human renal proximal tubular cell line HK-2. AKI was induced in mice and HK-2 cells using ischemia-reperfusion injury and hypoxia, respectively. Mitochondrial division inhibitor 1, an inhibitor of DRP1 (dynamin-related protein 1), was used to reduce mitochondrial fragmentation. Gene and protein expression were then examined to assess inflammation and fibrosis. The results of bioenergetic studies for mitochondrial function, oxidative stress, and angiogenesis were assessed. RESULTS: LTBP4 expression was upregulated in the renal tissues of patients with AKI. Ltbp4-knockdown mice showed increased renal tissue injury and mitochondrial fragmentation after ischemia-reperfusion injury, as well as increased inflammation, oxidative stress, and fibrosis, and decreased angiogenesis. in vitro studies using HK-2 cells revealed similar results. The energy profiles of Ltbp4-deficient mice and LTBP4-deficient HK-2 cells indicated decreased ATP production. LTBP4-deficient HK-2 cells exhibited decreased mitochondrial respiration and glycolysis. Human aortic endothelial cells and human umbilical vein endothelial cells exhibited decreased angiogenesis when treated with LTBP4-knockdown conditioned media. Mitochondrial division inhibitor 1 treatment ameliorated inflammation, oxidative stress, and fibrosis in mice and decreased inflammation and oxidative stress in HK-2 cells. CONCLUSIONS: Our study is the first to demonstrate that LTBP4 deficiency increases AKI severity, consequently leading to chronic kidney disease. Potential therapies focusing on LTBP4-associated angiogenesis and LTBP4-regulated DRP1-dependent mitochondrial division are relevant to renal injury.

Lipid-Based Nanocarriers in Renal RNA Therapy.

Kidney disease is a multifactorial problem, with a growing prevalence and an increasing global burden. With the latest worldwide data suggesting that chronic kidney disease (CKD) is the 12th leading cause of death, it is no surprise that CKD remains a public health problem that requires urgent attention. Multiple factors contribute to kidney disease, each with its own pathophysiology and pathogenesis. Furthermore, microRNAs (miRNAs) have been linked to several types of kidney diseases. As dysregulation of miRNAs is often seen in some diseases, there is potential in the exploitation of this for therapeutic applications. In addition, uptake of interference RNA has been shown to be rapid in kidneys making them a good candidate for RNA therapy. The latest advancements in RNA therapy and lipid-based nanocarriers have enhanced the effectiveness and efficiency of RNA-related drugs, thereby making RNA therapy a viable treatment option for renal disease. This is especially useful for renal diseases, for which a suitable treatment is not yet available. Moreover, the high adaptability of RNA therapy combined with the low risk of lipid-based nanocarriers make for an attractive treatment choice. Currently, there are only a small number of RNA-based drugs related to renal parenchymal disease, most of which are in different stages of clinical trials. We propose the use of miRNAs or short interfering RNAs coupled with a lipid-based nanocarrier as a delivery vehicle for managing renal disease.

LTBP4 affects renal fibrosis by influencing angiogenesis and altering mitochondrial structure.

Transforming growth factor beta (TGFß) signalling regulates extracellular matrix accumulation known to be essential for the pathogenesis of renal fibrosis; latent transforming growth factor beta binding protein 4 (LTBP4) is an important regulator of TGFß activity. To date, the regulation of LTBP4 in renal fibrosis remains unknown. Herein, we report that LTBP4 is upregulated in patients with chronic kidney disease and fibrotic mice kidneys created by unilateral ureteral obstruction (UUO). Mice lacking the short LTBP4 isoform (Ltbp4S-/-) exhibited aggravated tubular interstitial fibrosis (TIF) after UUO, indicating that LTBP4 potentially protects against TIF. Transcriptomic analysis of human proximal tubule cells overexpressing LTBP4 revealed that LTBP4 influences angiogenic pathways; moreover, these cells preserved better mitochondrial respiratory functions and expressed higher vascular endothelial growth factor A (VEGFA) compared to wild-type cells under hypoxia. Results of the tube formation assay revealed that additional LTBP4 in human umbilical vein endothelial cell supernatant stimulates angiogenesis with upregulated vascular endothelial growth factor receptors (VEGFRs). In vivo, aberrant angiogenesis, abnormal mitochondrial morphology and enhanced oxidative stress were observed in Ltbp4S-/- mice after UUO. These results reveal novel molecular functions of LTBP4 stimulating angiogenesis and potentially impacting mitochondrial structure and function. Collectively, our findings indicate that LTBP4 protects against disease progression and may be of therapeutic use in renal fibrosis.

Visualizing tRNA-dependent mistranslation in human cells.

High-fidelity translation and a strictly accurate proteome were originally assumed as essential to life and cellular viability. Yet recent studies in bacteria and eukaryotic model organisms suggest that proteome-wide mistranslation can provide selective advantages and is tolerated in the cell at higher levels than previously thought (one error in 6.9 × 10-4 in yeast) with a limited impact on phenotype. Previously, we selected a tRNAPro containing a single mutation that induces mistranslation with alanine at proline codons in yeast. Yeast tolerate the mistranslation by inducing a heat-shock response and through the action of the proteasome. Here we found a homologous human tRNAPro (G3:U70) mutant that is not aminoacylated with proline, but is an efficient alanine acceptor. In live human cells, we visualized mistranslation using a green fluorescent protein reporter that fluoresces in response to mistranslation at proline codons. In agreement with measurements in yeast, quantitation based on the GFP reporter suggested a mistranslation rate of up to 2-5% in HEK 293 cells. Our findings suggest a stress-dependent phenomenon where mistranslation levels increased during nutrient starvation. Human cells did not mount a detectable heat-shock response and tolerated this level of mistranslation without apparent impact on cell viability. Because humans encode ∼600 tRNA genes and the natural population has greater tRNA sequence diversity than previously appreciated, our data also demonstrate a cell-based screen with the potential to elucidate mutations in tRNAs that may contribute to or alleviate disease.

The Caenorhabditis elegans Female-Like State: Decoupling the Transcriptomic Effects of Aging and Sperm Status.

Understanding genome and gene function in a whole organism requires us to fully comprehend the life cycle and the physiology of the organism in question. Caenorhabditis elegans XX animals are hermaphrodites that exhaust their sperm after 3 d of egg-laying. Even though C. elegans can live for many days after cessation of egg-laying, the molecular physiology of this state has not been as intensely studied as other parts of the life cycle, despite documented changes in behavior and metabolism. To study the effects of sperm depletion and aging of C. elegans during the first 6 d of adulthood, we measured the transcriptomes of first-day adult hermaphrodites and sixth-day sperm-depleted adults, and, at the same time points, mutant fog-2(lf) worms that have a feminized germline phenotype. We found that we could separate the effects of biological aging from sperm depletion. For a large subset of genes, young adult fog-2(lf) animals had the same gene expression changes as sperm-depleted sixth-day wild-type hermaphrodites, and these genes did not change expression when fog-2(lf) females reached the sixth day of adulthood. Taken together, this indicates that changing sperm status causes a change in the internal state of the worm, which we call the female-like state. Our data provide a high-quality picture of the changes that happen in global gene expression throughout the period of early aging in the worm.

Communication between oocytes and somatic cells regulates volatile pheromone production in Caenorhabditis elegans.

Males of the androdioecious species Caenorhabditis elegans are more likely to attempt to mate with and successfully inseminate C. elegans hermaphrodites that do not concurrently harbor sperm. Although a small number of genes have been implicated in this effect, the mechanism by which it arises remains unknown. In the context of the battle of the sexes, it is also unknown whether this effect is to the benefit of the male, the hermaphrodite, or both. We report that successful contact between mature sperm and oocyte in the C. elegans gonad at the start of fertilization causes the oocyte to release a signal that is transmitted to somatic cells in its mother, with the ultimate effect of reducing her attractiveness to males. Changes in hermaphrodite attractiveness are tied to the production of a volatile pheromone, the first such pheromone described in C. elegans.

A comparison of applanation tonometry using conventional reusable goldmann prisms and disposable prisms.

PURPOSE: To determine the agreement between intraocular pressure (IOP) measurements using conventional Goldmann applanation tonometry (GAT) and Tonosafe disposable prisms, and also to provide a comprehensive cost analysis of the use of both types of prisms METHODS: : In this prospective observational study, 198 eyes of 100 glaucoma patients had their IOPs measured by 5 consultant ophthalmologists. Data were analyzed using the Bland-Altman method of differences, and correlation was measured using the Pearson coefficient. An analysis of the cost incurred using the 2 methods over a 6-month period was performed. RESULTS: The majority were Chinese (82%), with a male preponderance (57%). The range of IOPs as measured by GAT was 4 to 34 mm Hg. Using the Bland-Altman method to compare GAT and disposable prisms, the bias was 0.2 mm Hg. Tonosafe overestimated the IOP by 0.2 mm Hg in the right eye and underestimated it by 0.2 mm Hg in the left eye. The Tonosafe IOP correlated well with GAT, with a Pearson coefficient of correlation(r) of 0.91 (P<0.0005) for the right eye and 0.92 (P<0.0005) for the left eye, respectively. For those with GAT IOP≥21 mm Hg (n=26), Tonosafe underestimated the IOP by 0.35 mm Hg. The cost incurred by Tonosafe prisms was approximately 8 times that of GAT, but the cost differential reverses when GAT had to be replaced after every 100 cycles of disinfection. CONCLUSIONS: We found a good correlation between Tonosafe prisms and conventional GAT in measuring the IOP. Tonosafe prisms may be of use, especially if the risk of transmission of infection is high. However, cost may limit its more widespread use.

Patient acceptance and attitude toward an alternative method of subconjunctival injection for the medical treatment of glaucoma.

PURPOSE: Nonadherence to glaucoma medications may be a major cause of treatment failure. We examined the acceptance of glaucoma patients toward a possible new route of administering glaucoma medication by subconjunctival injection. PATIENTS AND METHODS: Patients were recruited from specialist glaucoma clinics on a voluntary basis. Trained interviewers administered a 30-item questionnaire and an information sheet with details of an alternative subconjunctival injection route involving injections at 3-month intervals. Outcome measures regarding acceptance of the new procedure, social situational factors, disease factors, and treatment factors were assessed. RESULTS: A total of 151 patients participated in this study. Of the 151 patients 112 (74.2%) were willing to have their glaucoma medication given by the new method of subconjunctival injection, 101 of 112 (90.2%) were willing to accept it at the same cost as their present medication, and 87 of 101 (86.1%) were willing to accept it even at a higher cost. These patients tended to be on a greater number of medications (P=0.006), and medicating more frequently in a day (P=0.003). Nine of 10 (90%) patients who were admitted to nonadherence were willing to accept subconjunctival injections at 3-month intervals in place of their topical medication. CONCLUSIONS: Our study found that 74% of glaucoma patients were willing to accept an alternative form of glaucoma treatment through 3-monthly subconjunctival injections. A large proportion of patients who were admitted to nonadherence to topical medication were willing to consider this alternative method of medication. Our findings are helpful when developing patient-acceptable drug-delivery regimes, which may alleviate the need for daily medication.

Central corneal thickness and its associations with ocular and systemic factors: the Singapore Malay Eye Study.

PURPOSE: To examine the association between central corneal thickness (CCT) and potential systemic and ocular factors affecting CCT in an Asian population. DESIGN: Population-based cross-sectional study. METHODS: A total of 3,280 (78.7% response) adults aged 40 to 80 years of Malay ethnicity living in Singapore underwent a standardized interview and ocular and systemic examination at a centralized study clinic. CCT was measured with an ultrasound pachymeter. Blood samples were obtained to determine serum glucose, cholesterol, and triglyceride levels, and urine samples to determine glomerular filtration rate. The presence of diabetes, hypertension, chronic kidney disease (CKD), and metabolic syndrome were defined based on a combination of investigation results and participant's history. RESULTS: CCT was obtained from 3,239 individuals. CCT was normally distributed with a mean of 541.2 microm in the right eye. While controlling for age and gender, CCT was greater in individuals with higher body mass index (BMI) (P = .038), greater intraocular pressure (IOP) (P < .001), greater axial length (P = .005), and greater radius of corneal curvature (P < .001). Individuals with CKD (P = 0.012) and metabolic syndrome (P < .001) also had greater CCT. CONCLUSION: CCT is associated with higher IOP, longer axial length, and greater radius of corneal curvature, as well as higher BMI, metabolic syndrome, and CKD.

Detection of multiple myeloma by PET/CT in a patient with colon cancer.

A 76-year-old man with previous sigmoid colon resection for adenocarcinoma had low back pain for 2 months. Whole-body bone scintigraphy showed multiple focal Tc-99m methylene diphosphonate (MDP)-avid lesions in both rib cages and 3 lumbar vertebrae, indicating metastases. F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) imaging was performed for further evaluation of this possible metastatic disease and demonstrated the lumbar and costal metastases and several hypermetabolic areas in the pelvic bones, multiple thoracic vertebrae, both shoulders, and the right femur. Histopathologic examination of the right-sided iliac crest, however, revealed multiple myeloma.

Diabetes, hyperglycemia, and central corneal thickness: the Singapore Malay Eye Study.

PURPOSE: To examine the relationship of diabetes and hyperglycemia with central corneal thickness (CCT) in Malay adults in Singapore. DESIGN: Population-based cross-sectional study. PARTICIPANTS: Three thousand two hundred eighty Malay adults ages 40-80 years living in Singapore. METHODS: The study population was selected using an age-stratified random sampling procedure of Malay 40- to 80-year-olds living in the southwestern part of Singapore. Participants had a standardized interview, examination, and ocular imaging at a centralized study clinic. Central corneal thickness was measured with an ultrasound pachymeter, and nonfasting serum glucose and glycosylated hemoglobin (Hb A(1C)) was obtained from all participants. Diabetes was defined as having nonfasting glucose levels of > or =200 mg/dl (11.1 mmol/l), a self-report of diabetic medication use, or physician diagnosis of diabetes. MAIN OUTCOME MEASURES: Central corneal thickness. RESULTS: Of the 3280 (78.7% response) participants, data on CCT were available on 3239 right eyes. Central corneal thickness was normally distributed, with a mean of 541.2 microm. There were 748 persons with diabetes (23.0%). After controlling for age and gender, central corneas were significantly thicker in persons with diabetes than in those without diabetes (547.2 microm vs. 539.3 microm, P<0.001) and, in the total population, with higher serum glucose (539.6, 540.2, 541.3, and 544.4, comparing increasing glucose quartiles; P = 0.023) and higher Hb A(1C) (537.8, 541.0, 541.4, and 545.5, comparing increasing Hb A(1C) quartiles; P<0.001) levels. In multiple linear regression models adjusting for age, intraocular pressure (IOP), body mass index, and axial length, persons with diabetes had, on average, central corneas 6.50 microm thicker than those of persons without diabetes. CONCLUSIONS: This population-based study among Malays showed that diabetes and hyperglycemia are associated with thicker central corneas, independent of age and IOP levels. These findings may have implications for understanding the relationship between diabetes and glaucoma.

Comparison of needlescopic appendectomy versus conventional laparoscopic appendectomy: a randomized controlled trial.

Laparoscopic appendectomy has been shown to improve postoperative recovery when compared with open appendectomy. The present randomized trial was conducted to evaluate any further difference in outcome between needlescopic appendectomy (NA) and conventional laparoscopic appendectomy (CLA) in the management of acute appendicitis. Patients with the clinical diagnosis of acute appendicitis were randomized to either NA (instrument size < or = 3 mm) or CLA (instrument size > or = 5 mm). Standardized anesthetic technique and perioperative management were adopted. The primary end point was length of postoperative hospital stay. Other parameters such as conversion rate, postoperative pain score and analgesic requirement, return of bowel function, resumption of normal activities, complication rate, and length of the final scars were also assessed and compared. A total of 363 patients (NA: 174, CLA: 189) were recruited. Both approaches could accurately arrive at the diagnosis (NA: 98.3%; CLA: 100%). Compared with CLA, NA resulted in a significantly longer operation time (P = 0.015) and a higher conversion rate (P < 0.001). The final scars of the NA group were significantly shorter when compared with the CLA group (P < 0.001). Otherwise, there was no statistical difference between the 2 groups in terms of complication rate, postoperative pain score, length of postoperative stay, and other recovery parameters. NA resulted in a longer operation time and higher conversion rate. Except for a smaller scar, the present study was unable to demonstrate any other short-term benefits. Thus, the technique cannot be routinely recommended.

Transmission of human papillomaviruses from mother to child.

Exfoliated cervical epithelial cells from women 6 weeks postpartum were analyzed for human papillomavirus (HPV) DNA using the polymerase chain reaction, and results were compared with those from buccal mucosal smears from their babies. Eleven mothers had genital genotypes of HPV in their cervical smears, and the children of 8 of these had HPV of the same genotype in buccal mucosal cell samples. Nineteen mothers had no HPV DNA detected in their cervical smears, and 1 of the buccal mucosal cell samples from their children was positive for HPV DNA (p < 0.0001). Contamination of a child's mouth with 'genital' HPV from a mother's cervix appears to occur commonly at birth or in the perinatal period, and to persist for at least 6 weeks. This observation has implications for the epidemiology and management of HPV associated cancer and precancerous conditions in the cervix and the mouth.