Detection of genomic mutations in blood and urine free circulating tumour DNA in patients with inoperable and metastatic lung adenocarcinoma harbouring an EGFR mutation in tissue: a UK pilot study.

The development of methodologies to analyse circulating tumour DNA (ctDNA) in the blood or urine of cancer patients provides an invaluable resource that can be used for diagnosis and prognosis and to evaluate response to treatments. Lung cancer has seen in the last years a revolution in treatment strategy with the use of several classes of EGFR inhibitors. However, almost invariably, resistance to such therapies appears. In this paper, we describe a pilot, longitudinal study with 20 patients with confirmed EGFR mutations in tissue biopsy for lung cancer. The objective of the study was to determine whether ctDNA from plasma and/or urine could be used to monitor the EGFR mutational status of patients with confirmed EGFR mutation-positive non-small cell lung cancer (NSCLC) during treatment with EGFR inhibitors. Blood and urine were collected monthly over periods ranging from 6 to 16 months. CtDNA was analysed in each patient for the presence of several known mutations that predispose to resistance to EGFR inhibitors. We have proven that serial monitoring of ctDNA from both plasma and urine is feasible and that patients are willing to participate in this process. We have also shown that longitudinal ctDNA monitoring may detect resistance mutations before the development of radiological and clinical disease progression.

Safety and efficacy of vitamin B in cancer treatments: A systematic review.

BACKGROUND: With rising rates of complementary and alternative medicine use, the exploration of complementary and alternative medicine integration into oncology treatments is becoming increasingly prevalent. Vitamin B compounds including B1, B2, B3, B5, B6, B9, and B12, have all been proposed as potentially beneficial in cancer prevention and treatment as well as side effect management; however, many studies contain contradicting evidence regarding the utility of B vitamins within oncology. Thus, the aim of this study was to evaluate the safety and efficacy of Vitamin B supplementation in the oncology setting. DESIGN: A systematic review was conducted following The Preferred Reporting Items for Systematic Reviews and Meta-Analyses-Scoping Reviews guidelines, using pre-specified search terms in PubMed to include randomized control trials, clinical trials, and case studies. Two reviewers independently reviewed titles, abstracts, and full-text articles for inclusion, with a third reviewer resolving conflicts, before the included articles underwent data extraction and quality appraisal. Data extraction was conducted through COVIDENCE, which was used to manage and track the data during the search process. RESULTS: Out of 694 articles initially identified, 25 articles met the inclusion criteria and were included in the review. Designs of the studies varied, including randomized control trials, clinical trials, and case/cohort studies. The impact of vitamin supplementation on cancer risk varied. Several studies found that certain B vitamin supplementation lowered cancer risk: B9 and B6 in nasopharyngeal carcinoma (n = 1200 patients) and in pancreatic cancer (n = 258 patients); B3 in hepatocellular carcinoma (n = 494,860 patients); B6 in breast cancer (n = 27,853 patients); and B9 in BRCA1-positive breast cancer (n = 400 patients). However, some studies found that certain B vitamin supplementation increased the risk or negative outcomes of cancer: B6 during nasopharyngeal carcinoma treatment (n = 592 patients); B6 in risk of hepatocellular carcinoma (n = 494,860 patients); and B9 plasma levels in breast cancer (n = 164 patients). Due to the many adverse effects that occur in cancer treatment, the effectiveness of Vitamin B supplementation in alleviating adverse effects was evaluated. In two separate studies, Vitamin B6 and Vitamin B12 supplementation with acupuncture was found to be effective as adjunct therapies aimed to reduce chemotherapy-induced peripheral neuropathy (n = 23 patients and n = 104 patients, respectively). No significant findings were established regarding B vitamin supplementation in chemotherapy-induced hand-foot syndrome. CONCLUSIONS: In this systematic review we concluded that B vitamin supplements have varying data regarding safety and efficacy in cancer. Taking into account the etiology of the cancer, the specific B-vitamin, and the presence of any side effects could help guide utilization of the data found in this review. Large, randomized controlled trials are needed to confirm these findings among various cancer diagnoses and stages. Given the widespread utilization of supplements, healthcare providers should understand the safety and efficacy of vitamin B supplementation to address questions that arise in caring for those with cancer.

A repositioning screen using an FGFR2 splicing reporter reveals compounds that regulate epithelial-mesenchymal transitions and inhibit growth of prostate cancer xenografts.

Research in the area of hallmarks of cancer has opened the possibility of designing new therapies based on modulating these cancer properties. We present here a screen designed to find chemicals that modulate epithelial-mesenchymal transitions (EMTs) in prostate cancer. For screening, we used a repurposing library and, as a readout, an FGFR2-based splicing reporter, which has been shown previously to be a sensor for EMTs. Various properties of cancer cells were assessed, signaling pathways investigated, and in vivo experiments in nude mice xenografts performed. The screen yielded three hit compounds (a T-type Ca channel inhibitor, an L-type Ca channel inhibitor, and an opioid antagonist) that switch FGFR2 splicing and induce an epithelial phenotype in prostate cancer cells. The compounds affected differently various properties of cancer cells, but all of them decreased cell migration, which is in line with modulating EMTs. We further present mechanistic insights into one of the compounds, nemadipine-A. The administration of nemadipine-A intraperitoneally in a nude mouse xenograft model of prostate cancer slowed tumor growth. To conclude, we show that knowledge of the molecular mechanisms that connect alternative splicing and various cancer properties may be used as a platform for drug development.

Reduced Glomerular Filtration in Diabetes Is Attributable to Loss of Density and Increased Resistance of Glomerular Endothelial Cell Fenestrations.

BACKGROUND: Glomerular endothelial cell (GEnC) fenestrations are recognized as an essential component of the glomerular filtration barrier, yet little is known about how they are regulated and their role in disease. METHODS: We comprehensively characterized GEnC fenestral and functional renal filtration changes including measurement of glomerular Kf and GFR in diabetic mice (BTBR ob-/ob- ). We also examined and compared human samples. We evaluated Eps homology domain protein-3 (EHD3) and its association with GEnC fenestrations in diabetes in disease samples and further explored its role as a potential regulator of fenestrations in an in vitro model of fenestration formation using b.End5 cells. RESULTS: Loss of GEnC fenestration density was associated with decreased filtration function in diabetic nephropathy. We identified increased diaphragmed fenestrations in diabetes, which are posited to increase resistance to filtration and further contribute to decreased GFR. We identified decreased glomerular EHD3 expression in diabetes, which was significantly correlated with decreased fenestration density. Reduced fenestrations in EHD3 knockdown b.End5 cells in vitro further suggested a mechanistic role for EHD3 in fenestration formation. CONCLUSIONS: This study demonstrates the critical role of GEnC fenestrations in renal filtration function and suggests EHD3 may be a key regulator, loss of which may contribute to declining glomerular filtration function through aberrant GEnC fenestration regulation. This points to EHD3 as a novel therapeutic target to restore filtration function in disease.

A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties.

Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3' splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A's terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3' splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.

Modulation of the Apoptosis Gene Bcl-x Function Through Alternative Splicing.

Apoptosis plays a vital role in cell homeostasis during development and disease. Bcl-x, a member of the Bcl-2 family of proteins, is a mitochondrial transmembrane protein that functions to regulate the intrinsic apoptosis pathway. An alternative splicing (AS) event in exon 2 of Bcl-x results in two isoforms of Bcl-x with antagonistic effects on cell survival: Bcl-xL (long isoform), which is anti-apoptotic, and Bcl-xS (short isoform), which is pro-apoptotic. Bcl-xL is the most abundant Bcl-x protein and functions to inhibit apoptosis by a number of different mechanisms including inhibition of Bax. In contrast, Bcl-xS can directly bind to and inhibit the anti-apoptotic Bcl-xL and Bcl-2 proteins, resulting in the release of the pro-apoptotic Bak. There are multiple splice factors and signaling pathways that influence the Bcl-xL/Bcl-xS splicing ratio, including serine/arginine-rich (SR) proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), transcription factors, and cytokines. Dysregulation of the AS of Bcl-x has been implicated in cancer and diabetes. In cancer, the upregulation of Bcl-xL expression in tumor cells can result in resistance to chemotherapeutic agents. On the other hand, dysregulation of Bcl-x AS to promote Bcl-xS expression has been shown to be detrimental to pancreatic ß-cells in diabetes, resulting in ß-cell apoptosis. Therefore, manipulation of the splice factor, transcription factor, and signaling pathways that modulate this splicing event is fast emerging as a therapeutic avenue in the treatment of cancer and diabetes.

The natural drug DIAVIT is protective in a type II mouse model of diabetic nephropathy.

There is evidence to suggest that abnormal angiogenesis, inflammation, and fibrosis drive diabetic nephropathy (DN). However, there is no specific treatment to counteract these processes. We aimed to determine whether DIAVIT, a natural Vaccinium myrtillus (blueberry) and Hippophae Rhamnoides (sea buckthorn) extract, is protective in a model of type II DN. Diabetic db/db mice were administered DIAVIT in their drinking water for 14 weeks. We assessed the functional, structural, and ultra-structural phenotype of three experimental groups (lean+vehicle, db/db+vehicle, db/db+DIAVIT). We also investigated the angiogenic and fibrotic pathways involved in the mechanism of action of DIAVIT. Diabetic db/db mice developed hyperglycaemia, albuminuria, and an increased glomerular water permeability; the latter two were prevented by DIAVIT. db/db mice developed fibrotic glomeruli, endothelial insult, and glomerular ultra-structural changes, which were not present in DIAVIT-treated mice. Vascular endothelial growth factor A (VEGF-A) splicing was altered in the db/db kidney cortex, increasing the pro-angiogenic VEGF-A165 relative to the anti-angiogenic VEGF-A165b. This was partially prevented with DIAVIT treatment. Delphinidin, an anthocyanin abundant in DIAVIT, increased the VEGF-A165b expression relative to total VEGF-A165 in cultured podocytes through phosphorylation of the splice factor SRSF6. DIAVIT, in particular delphinidin, alters VEGF-A splicing in type II DN, rescuing the DN phenotype. This study highlights the therapeutic potential of natural drugs in DN through the manipulation of gene splicing and expression.

Modulation of Receptor Tyrosine Kinase Activity through Alternative Splicing of Ligands and Receptors in the VEGF-A/VEGFR Axis.

Vascular endothelial growth factor A (VEGF-A) signaling is essential for physiological and pathological angiogenesis. Alternative splicing of the VEGF-A pre-mRNA gives rise to a pro-angiogenic family of isoforms with a differing number of amino acids (VEGF-Axxxa), as well as a family of isoforms with anti-angiogenic properties (VEGF-Axxxb). The biological functions of VEGF-A proteins are mediated by a family of cognate protein tyrosine kinase receptors, known as the VEGF receptors (VEGFRs). VEGF-A binds to both VEGFR-1, largely suggested to function as a decoy receptor, and VEGFR-2, the predominant signaling receptor. Both VEGFR-1 and VEGFR-2 can also be alternatively spliced to generate soluble isoforms (sVEGFR-1/sVEGFR-2). The disruption of the splicing of just one of these genes can result in changes to the entire VEGF-A/VEGFR signaling axis, such as the increase in VEGF-A165a relative to VEGF-A165b resulting in increased VEGFR-2 signaling and aberrant angiogenesis in cancer. Research into this signaling axis has recently focused on manipulating the splicing of these genes as a potential therapeutic avenue in disease. Therefore, further research into understanding the mechanisms by which the splicing of VEGF-A/VEGFR-1/VEGFR-2 is regulated will help in the development of drugs aimed at manipulating splicing or inhibiting specific splice isoforms in a therapeutic manner.

Anti-angiogenic isoform of vascular endothelial growth factor-A in cardiovascular and renal disease.

Accumulating evidence suggests that pathologic interactions between the heart and the kidney can contribute to the progressive dysfunction of both organs. Recently, there has been an increase in the prevalence of cardiovascular disease (CVD) and chronic kidney disease (CKD) due to increasing obesity rates. It has been reported that obesity causes various heart and renal disorders and appears to accelerate their progression. Vascular endothelial growth factor-A (VEGF-A) is a major regulator of angiogenesis and vessel permeability, and is associated with CVD and CKD. It is now recognized that alternative VEGF-A gene splicing generates VEGF-A isoforms that differ in their biological actions. Proximal splicing that includes an exon 8a sequence results in pro-angiogenic VEGF-A165a, whereas distal splicing inclusive of exon 8b yields the anti-angiogenic isoform of VEGF-A (VEGF-A165b). This review highlights several recent preclinical and clinical studies on the role of VEGF-A165b in CVD and CKD as a novel function of VEGF-A. This review also discusses potential therapeutic approaches of the use of VEGF-A in clinical settings as a potential circulating biomarker for CVD and CKD.

Assessment of Kidney Function in Mouse Models of Glomerular Disease.

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

Modulation of VEGF-A Alternative Splicing as a Novel Treatment in Chronic Kidney Disease.

Vascular endothelial growth factor A (VEGF-A) is a prominent pro-angiogenic and pro-permeability factor in the kidney. Alternative splicing of the terminal exon of VEGF-A through the use of an alternative 3' splice site gives rise to a functionally different family of isoforms, termed VEGF-Axxxb, known to have anti-angiogenic and anti-permeability properties. Dysregulation of the VEGF-Axxx/VEGF-Axxxb isoform balance has recently been reported in several kidney pathologies, including diabetic nephropathy (DN) and Denys-Drash syndrome. Using mouse models of kidney disease where the VEGF-A isoform balance is disrupted, several reports have shown that VEGF-A165b treatment/over-expression in the kidney is therapeutically beneficial. Furthermore, inhibition of certain splice factor kinases involved in the regulation of VEGF-A terminal exon splicing has provided some mechanistic insight into how VEGF-A splicing could be regulated in the kidney. This review highlights the importance of further investigation into the novel area of VEGF-A splicing in chronic kidney disease pathogenesis and how future studies may allow for the development of splicing-modifying therapeutic drugs.

Vascular Endothelial Growth Factor-A165b Restores Normal Glomerular Water Permeability in a Diphtheria-Toxin Mouse Model of Glomerular Injury.

BACKGROUND/AIMS: Genetic cell ablation using the human diphtheria toxin receptor (hDTR) is a new strategy used for analysing cellular function. Diphtheria toxin (DT) is a cytotoxic protein that leaves mouse cells relatively unaffected, but upon binding to hDTR it ultimately leads to cell death. We used a podocyte-specific hDTR expressing (Pod-DTR) mouse to assess the anti-permeability and cyto-protective effects of the splice isoform vascular endothelial growth factor (VEGF-A165b). METHODS: The Pod-DTR mouse was crossed with a mouse that over-expressed VEGF-A165b specifically in the podocytes (Neph-VEGF-A165b). Wild type (WT), Pod-DTR, Neph-VEGF-A165b and Pod-DTR X Neph-VEGF-A165b mice were treated with several doses of DT (1, 5, 100, and 1,000 ng/g bodyweight). Urine was collected and the glomerular water permeability (LpA/Vi) was measured ex vivo after 14 days. Structural analysis and podocyte marker expression were also assessed. RESULTS: Pod-DTR mice developed an increased glomerular LpA/Vi 14 days after administration of DT (all doses), which was prevented when the mice over-expressed VEGF-A165b. No major structural abnormalities, podocyte ablation or albuminuria was observed in Pod-DTR mice, indicating this to be a mild model of podocyte disease. However, a change in expression and localisation of nephrin within the podocytes was observed, indicating disruption of the slit diaphragm in the Pod-DTR mice. This was prevented in the Pod-DTR X Neph-VEGF-A165b mice. CONCLUSION: Although only a mild model of podocyte injury, over-expression of the anti-permeability VEGF-A165b isoform in the podocytes of Pod-DTR mice had a protective effect. Therefore, this study further highlights the therapeutic potential of VEGF-A165b in glomerular disease.

Risky business: Changes in boldness behavior in male Siamese fighting fish, Betta splendens, following exposure to an antiandrogen.

Components of boldness, such as activity level and locomotion, influence an individual's ability to avoid predators and acquire resources, generating fitness consequences. The presence of endocrine disrupting chemicals (EDCs) in the aquatic environment may affect fitness by changing morphology or altering behaviors like courtship and exploration. Most research on EDC-generated behavioral effects has focused on estrogen mimics and reproductive endpoints. Far fewer studies have examined the effects of other types of EDCs or measured non-reproductive behaviors. EDCs with antiandrogenic properties are present in waterways yet we know little about their effects on exposed individuals although they may produce effects similar to those caused by estrogen mimics because they act on the same hormonal pathway. To examine the effects of antiandrogens on boldness, this study exposed male Siamese fighting fish, Betta splendens, to a high or low dose of one of two antiandrogens, vinclozolin or flutamide, and observed behavior in three boldness assays, both before and after exposure. Overall, antiandrogen exposure increased boldness behavior, especially following exposure to the higher dose. Whether or not antiandrogen exposure influenced boldness, as well as the nature and intensity of the effect, was assay-dependent. This demonstrates the importance of studying EDC effects in a range of contexts and, at least within this species, suggests that antiandrogenic compounds may generate distinct physiological effects in different situations. How and why the behavioral effects differ from those caused by exposure to an estrogen mimic, as well as the potential consequences of increased activity levels, are discussed. Exposure to an antiandrogen, regardless of dose, produced elevated activity levels and altered shoaling and exploration in male Siamese fighting fish. These modifications may have fitness consequences.

A multidisciplinary approach to laparoscopic sleeve gastrectomy among multiethnic adolescents in the United States.

BACKGROUND: Childhood obesity has become a serious public health problem in our country with a prevalence that is disproportionately higher among minority groups. Laparoscopic sleeve gastrectomy (LSG) is gaining attention as a safe bariatric alternative for severely obese adolescents. STUDY DESIGN: A retrospective study on morbidly obese adolescents that underwent LSG at our institution from 2009 to 2017. Primary outcomes were weight loss as measured by change in BMI and percent excess weight loss (%EWL) at 1 year after surgery, resolution of comorbidities and occurrence of complications. RESULTS: Thirty-eight patients, of whom 71% were female and 74% were ethnic minorities, underwent LSG between 2009 and 2016. Mean age was 16.8years, mean weight was 132.0kg and mean BMI was 46.7. There were no surgical complications. Mean %EWL was 19.4%, 27.9%, 37.4%, 44.9%, and 47.7% at 1.5, 3, 6, 9, and 12month follow up visits, respectively. Comorbidity resolution rates were 100% for hypertension and nonalcoholic fatty liver disease, 91% for diabetes, 44% for prediabetes, 82% for dyslipidemia and 89% for OSA. CONCLUSIONS: LSG is an effective and safe method of treatment of morbid obesity in adolescents as it can significantly decrease excess body weight and resolve comorbid conditions. Further studies are needed to investigate the long-term effects of LSG in adolescents. CLINICAL RESEARCH STUDY: Descriptive case series with prospective database. LEVEL OF EVIDENCE: IV.

VEGF-A165 b protects against proteinuria in a mouse model with progressive depletion of all endogenous VEGF-A splice isoforms from the kidney.

KEY POINTS: Progressive depletion of all vascular endothelial growth factor A (VEGF-A) splice isoforms from the kidney results in proteinuria and increased glomerular water permeability, which are both rescued by over-expression of VEGF-A165 b only. VEGF-A165 b rescues the increase in glomerular basement membrane and podocyte slit width, as well as the decrease in sub-podocyte space coverage, produced by VEGF-A depletion. VEGF-A165 b restores the expression of platelet endothelial cell adhesion molecule in glomerular endothelial cells and glomerular capillary circumference. VEGF-A165 b has opposite effects to VEGF-A165 on the expression of genes involved in endothelial cell migration and proliferation. ABSTRACT: Chronic kidney disease is strongly associated with a decrease in the expression of vascular endothelial growth factor A (VEGF-A). However, little is known about the contribution of VEGF-A splice isoforms to kidney physiology and pathology. Previous studies suggest that the splice isoform VEGF-A165 b (resulting from alternative usage of a 3' splice site in the terminal exon) is protective for kidney function. In the present study, we show, in a quad-transgenic model, that over-expression of VEGF-A165 b alone is sufficient to rescue the increase in proteinuria, as well as glomerular water permeability, in the context of progressive depletion of all VEGF-A isoforms from the podocytes. Ultrastructural studies show that the glomerular basement membrane is thickened, podocyte slit width is increased and sub-podocyte space coverage is reduced when VEGF-A is depleted, all of which are rescued in VEGF-A165 b over-expressors. VEGF-A165 b restores the expression of platelet endothelial cell adhesion molecule-1 in glomerular endothelial cells and glomerular capillary circumference. Mechanistically, it increases VEGF receptor 2 expression both in vivo and in vitro and down-regulates genes involved in migration and proliferation of endothelial cells, otherwise up-regulated by the canonical isoform VEGF-A165 . The results of the present study indicate that manipulation of VEGF-A splice isoforms could be a novel therapeutic avenue in chronic glomerular disease.

Regional block via continuous caudal infusion as sole anesthetic for inguinal hernia repair in conscious neonates.

PURPOSE: The use of general anesthesia in young children has come under increasing scrutiny due to its potential long-term neurotoxic effects. Meanwhile, regional anesthesia for surgical procedures in neonates has many advantages, including preservation of respiratory status and faster return to feeding. We describe the successful use of 3% 2-chloroprocaine administered via continuous caudal infusion as the sole anesthetic agent during elective surgical procedures in infants. METHODS: A retrospective chart review of all patients who underwent elective surgical procedures under continuous caudal regional anesthetic at a single institution was performed. Thirty patients (27 males, three females) were identified: 28 patients underwent inguinal hernia repairs. Caudal anesthesia was established via continuous infusion of 3% 2-chloroprocaine through an indwelling catheter. RESULTS: Successful analgesia by regional block alone was achieved in all patients for the duration of each surgical procedure without need for rescue anesthesia. Mean operative time was 49 min. Patients were able to return to feeding immediately after surgery and were ready for discharge home within that day. CONCLUSION: Continuous caudal infusion of chloroprocaine is a safe and effective way to maintain adequate analgesia for elective surgeries in infants. This successful regional approach obviates the use of general anesthetic which reduces post-operative recovery time and avoids concerns for neurotoxicity.

Alternative Splicing in CKD.

Alternative splicing (AS) has emerged in the postgenomic era as one of the main drivers of proteome diversity, with ≥94% of multiexon genes alternatively spliced in humans. AS is therefore one of the main control mechanisms for cell phenotype, and is a process deregulated in disease. Numerous reports describe pathogenic mutations in splice factors, splice sites, or regulatory sequences. Additionally, compared with the physiologic state, disease often associates with an abnormal proportion of splice isoforms (or novel isoforms), without an apparent driver mutation. It is therefore essential to study how AS is regulated in physiology, how it contributes to pathogenesis, and whether we can manipulate faulty splicing for therapeutic advantage. Although the disease most commonly linked to deregulation of AS in several genes is cancer, many reports detail pathogenic splice variants in diseases ranging from neuromuscular disorders to diabetes or cardiomyopathies. A plethora of splice variants have been implicated in CKDs as well. In this review, we describe examples of these CKD-associated splice variants and ideas on how to manipulate them for therapeutic benefit.

Initial results of endoscopic gastrocutaneous fistula closure in children using an over-the-scope clip.

Gastrocutaneous fistula (GCF) occurs commonly in pediatric patients after removal of long-term gastrostomy tubes. Although open repair is generally successful, endoscopic approaches may offer benefits in terms of incisional complications, postoperative pain, and procedure time. In addition, endoscopic approaches may offer particular benefit in patients with varied degrees of skin irritation or erosion surrounding a GCF, making surgical repair difficult, or patients with significant comorbidities, making minimal intervention and anesthesia time preferable. Over-the-scope (OSC) clips are a new technology that enables endoscopic closure of intestinal fistulas up to 2 cm in diameter. Six pediatric patients underwent endoscopic GCF closure using OSC clips under Institutional Review Board approval. The procedure was technically successful in 5 of 6 cases with an average operating time of 29 minutes. The technical failure required an open revision, whereas all other patients reported full healing of the GCF site at 1 month. All successful cases were performed as outpatients without postoperative narcotics. In addition, all patients reported high satisfaction with the procedure and cosmetic results. Endoscopic GCF closure using an OSC clip is technically feasible in the pediatric population. Based on limited cases with a 1-month follow-up, the functional and cosmetic results of technically successful cases are excellent. Endoscopic GCF closure is a potential alternative to standard surgical closure in patients with skin irritation or erosion and/or significant comorbidities.

Vascular Endothelial Growth Factor-A165b Is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy.

Diabetic nephropathy is the leading cause of ESRD in high-income countries and a growing problem across the world. Vascular endothelial growth factor-A (VEGF-A) is thought to be a critical mediator of vascular dysfunction in diabetic nephropathy, yet VEGF-A knockout and overexpression of angiogenic VEGF-A isoforms each worsen diabetic nephropathy. We examined the vasculoprotective effects of the VEGF-A isoform VEGF-A165b in diabetic nephropathy. Renal expression of VEGF-A165b mRNA was upregulated in diabetic individuals with well preserved kidney function, but not in those with progressive disease. Reproducing this VEGF-A165b upregulation in mouse podocytes in vivo prevented functional and histologic abnormalities in diabetic nephropathy. Biweekly systemic injections of recombinant human VEGF-A165b reduced features of diabetic nephropathy when initiated during early or advanced nephropathy in a model of type 1 diabetes and when initiated during early nephropathy in a model of type 2 diabetes. VEGF-A165b normalized glomerular permeability through phosphorylation of VEGF receptor 2 in glomerular endothelial cells, and reversed diabetes-induced damage to the glomerular endothelial glycocalyx. VEGF-A165b also improved the permeability function of isolated diabetic human glomeruli. These results show that VEGF-A165b acts via the endothelium to protect blood vessels and ameliorate diabetic nephropathy.